Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation

ABSTRACT

Disclosed are novel proteins, referred to as megakaryocyte growth and development factors (MGDFs; also generally referred to as Mpl ligands or thrombopoietin, that have a biological activity of stimulating the growth of megakaryocytes and augmenting the differentiation or maturation of megakaryocytes, ultimately to result in the production of platelets. MGDF derivatives comprising MGDF molecules attached to water soluble polymers, such as polyethylene glycol, are also disclosed, along with methods for their preparation. Also disclosed are processes for obtaining the MGDFs in homogeneous form from natural sources and producing them by recombinant genetic engineering techniques from mammals, including humans.

This application is a continuation-in-part of application Ser. No.08/252,628, filed May 31, 1994, which is a continuation-in part ofapplication Ser. No. 08/221,768, filed Mar. 31, 1994, abandoned.

FIELD OF THE INVENTION

The present invention relates to novel proteins, referred to hereinsynonymously as Mpl ligands or MGDFs, that stimulate the growth ofmegakaryocytes and augment the differentiation or maturation ofmegakaryocytes, with the ultimate effect of increasing the numbers ofplatelets. Also provided are processes for obtaining the proteins inhomogeneous form from natural sources and producing them by recombinantgenetic engineering techniques.

In another aspect, the present invention broadly relates to a novelclass of MGDF derivatives wherein an MGDF molecule is attached to awater soluble polymer, and methods for preparing such molecules. In yetanother aspect, the present invention relates to MGDF derivativeswherein an MGDF molecule is attached to one or more polyethylene glycol("PEG") groups, and methods of their preparation.

BACKGROUND OF THE INVENTION

At least two broad areas of research are involved in the presentinvention. The first relates to the development of megakaryocytes andsubsequent production of platelets, and the second relates to apolypeptide member of a growth factor receptor family, referred toherein as the Mpl receptor, and ligands thereof. Each of these areas ofresearch will now be outlined in the following.

A. Platelet Production from Megakaryocytes

Blood platelets are circulating cells that are crucial for theprevention of bleeding and for blood coagulation. Megakaryocytes are thecellular source of platelets and arise from a common bone marrowprecursor cell which gives rise to all hematopoietic cell lineages. Thiscommon precursor cell is known as the pluripotent stem cell or PPSC.

A hierarchy of megakaryocytic progenitor cells has been defined based onthe time of appearance and size of megakaryocyte (MK) colonies appearingin in vitro culture systems in response to appropriate growth factors.The burst-forming unit megakaryocyte (BFU-MK) is the most primitivemegakaryocyte progenitor cell. BFU-MK are thought ultimately to producenumerous colony forming unit megakaryocytes (CFU-MK), which are moredifferentiated MK progenitor cells.

As the MK cells undergo subsequent differentiation, they lose theability to undergo mitosis but acquire an ability to endoreduplicate.Endoreduplication (or endomitosis) is the phenomenon in cells of nucleardivision in the absence of cell division. Endoreduplication ultimatelyresults in an MK which is polyploid. Further MK maturation results inacquisition of cytoplasmic organelles and membrane constituents thatcharacterize platelets.

Platelets are produced from mature MK's by a poorly defined process thathas been suggested to be a consequence of MK physical fragmentation, orother mechanisms. Observations of extensive membranous structures withinmegakaryocytes has led to a model of platelet formation in which ademarcation membrane system outlines nascent platelets within the cellbody. Another model of platelet formation has developed fromobservations that megakaryocytes will form long cytoplasmic processesconstricted at platelet-sized intervals from which platelets presumablybreak off due to blood flow pressures in the marrow and/or in the lung.These cytoplasmic processes were termed proplatelets by Becker andDeBruyn to reflect their presumed precursor role in platelet formation.See Becker and DeBruyn, Amer. J. Anat. 145: 183 (1976).

FIG. 1 presents an overview of the various precursor cells involved inmegakaryocyte and platelet development. The cell at the far left-handside of the figure represents a PPSC, and the additional cells to theright of the PPSC in the figure represent BFU-MK, followed by CFU-MK.The cell that is undergoing endoreduplication, which is locatedimmediately to the right of the PPSC in the figure, is a maturemegakaryocyte cell. As a result of endomitosis, this cell has becomepolyploid. The next structure to the right includes long cytoplasmicprocesses emerging from the polyploid nucleus of the maturemegakaryocyte cell. In the far right-hand side of the figure are shown anumber of platelets that have been produced by fragmentation of thecytoplasmic processes.

The following is a summary of some prior publications relating to theabove description of megakaryocyte maturation and the production ofplatelets:

1. Williams, N. and Levine, R. F., British Journal of Haematology 52:173-180 (1982).

2. Levin, J., Molecular Biology and Differentiation of Megakaryocytes,pub. Wiley-Liss, Inc.: 1-10 (1990).

3. Gewirtz, A. M., The Biology of Hematopoiesis, pub. Wiley-Liss, Inc.:123-132 (1990).

4. Han, Z. C., et al., Int. J. Hematol. 54: 3-14 (1991).

5. Nieuwenhuis, H. K. and Sixma, J., New Eng. J. of Med. 327: 1812-1813(1992).

6. Long, M., Stem Cells 11: 33-40 (1993).

B. Regulation of Platelet Formation

A large body of data generated in many laboratories indicates thatplatelet production is regulated by humoral factors. The complexity ofthis biological process was not originally appreciated and currently itappears that a number of human growth factors possess this capability.

Megakaryocyte regulation occurs at multiple cellular levels. A number ofcytokines enhance platelet production by expanding the progenitor cellpool. A second group of humoral growth factors serves as maturationfactors acting on more differentiated cells to promoteendoreduplication. In addition, there appear to be two independentbiofeedback loops regulating these processes.

Several lineage nonspecific hematopoietic growth factors exert importanteffects on MK maturation. Granulocyte-macrophage colony stimulatingfactor (GM-CSF), interleukin-3 (IL-3), IL-6, IL-11, leukemia inhibitoryfactor (LIF), and erythropoietin (EPO) each individually promote humanMK maturation in vitro as determined by their effects on MK size,number, or ploidy. The MK maturational effects of LIF, IL-6, and IL-11are either partially (LIF and IL-6) or totally (IL-11) additive to thoseof IL-3. Such data from these prior publications suggested thatcombinations of cytokines may be necessary to promote MK maturation invivo.

The following is a summary of some prior publications relating to theregulation of megakaryocyte and platelet production:

7. Hoffman, R. et al., Blood Cells 13: 75-86 (1987).

8. Murphy, M. J., Hematology/Oncology Clinics of North America 3 (3):465-478 (1988).

9. Hoffman, R., Blood 74 (4): 1196-1212 (1989).

10. Mazur, E. M. and Cohen, J. L., Clin. Pharmacol. Ther., 46 (3):250-256 (1989).

11. Gewirtz, A. M. and Calabretta, B., Int. J. Cell Cloning 8: 267-276(1990).

12. Williams, N., Progress in Growth Factor Research 2: 81-95 (1990).

13. Gordon, M. S. and Hoffman, R., Blood 80 (2): 302-307 (1992).

14. Hunt, P. et al., Exp. Hematol. 21: 372-281 (1993).

15. Hunt, P. et al., Exp. Hematol. 21: 1295-1304 (1993).

It has also been reported (see reference 16) that human aplastic serumcontains a megakaryocyte colony stimulating activity distinct from IL-3,granulocyte colony stimulating factor, and factors present inlymphocyte-conditioned medium. However, the molecule responsible forthis activity was neither isolated nor characterized in the prior art.16. Mazur, E. M., et al., Blood 76: 290-297 (1990).

C. The Mpl Receptor

The myeloproliferative leukemia virus (MPLV) is a murinereplication-defective retrovirus that causes acute leukemia in infectedmammals. It has been discovered that a gene expressed by MPLV consistsof a part of the gene that encodes the retroviral envelope (or externalprotein coat) of the virus fused to a sequence that is related to thecytokine receptor family, including the receptors for GM-CSF, G-CSF, andEPO.

Expression of the MPLV gene described above has the interestingbiological property of causing murine progenitor cells of various typesto immediately acquire growth factor independence for both proliferationand terminal maturation. Moreover, some cultures of bone marrow cellsacutely transformed by MPLV contained megakaryocytes, suggesting aconnection between the MPLV gene and megakaryocyte growth anddifferentiation.

It is now recognized that the MPLV viral gene (referred to as v-Mpl) hasa homolog in mammalian cells, which is referred to as a cellular Mplgene (or c-Mpl). Using v-Mpl-derived probes, a cDNA corresponding to thehuman c-Mpl gene was cloned. See PCT published application WO 92/07074(published Apr. 30, 1992; discussed below). Sequence analysis has shownthat the protein encoded by the c-Mpl gene product belongs to the highlyconserved cytokine receptor superfamily, just like the homologous v-Mplgene product.

This cellular gene, c-Mpl, is thought to play a functional role inhematopoiesis based on the observation that its expression was found inbone marrow, spleen, and fetal liver from normal mice by RNAse probeprotection and RT-PCR experiments, but not in other tissues. Inparticular, c-Mpl is expressed on megakaryocytes. It has also beendemonstrated that the human cellular gene, human c-Mpl, is expressed inCD34 positive cells, including purified megakaryocytes and platelets.CD34 is an antigen that is indicative of early hematopoietic progenitorcells. Furthermore, exposure of CD34 positive cells to syntheticoligodeoxynucleotides that are anti-sense to the c-Mpl mRNA or messagesignificantly inhibits the colony forming ability of CFU-MKmegakaryocyte progenitors, but has no effect on erythroid orgranulomacrophage progenitors.

The above data and observations suggest that c-Mpl encodes a cellsurface molecule, referred to herein as the Mpl receptor, which binds toa ligand, that activates the receptor, possibly leading to productionand/or development of megakaryocytes.

PCT patent publication WO 92/07074 is directed to the sequence of theprotein produced by the c-Mpl gene, from both human and murine sources.This gene product, which is thought to be a receptor as explained above,is made up of at least three general regions or domains: anextracellular domain, a transmembrane domain, and an intracellular (orcytoplasmic) domain. Attached together, these domains make up the intactMpl receptor. This PCT publication also refers to a soluble form of thereceptor that substantially corresponds to the extracellular domain ofthe mature c-Mpl protein. The intracellular domain contains ahydrophobic region that, when attached via the transmembrane region tothe extracellular domain of the protein, renders the overall proteinsubject to aggregation and insolubility. On the other hand, when theextracellular domain of the c-Mpl gene product is separated from thetransmembrane domain and the intracellular domain, it becomes soluble,hence the extracellular form of the protein is referred to as a"soluble" form of the receptor.

The following is a summary of some prior publications relating to theabove description of the v-Mpl and c-Mpl receptors and genes:

17. Wendling, F., et al., Leukemia 3 (7): 475-480 (1989).

18. Wendling, F., et al., Blood 73 (5): 1161-1167 (1989).

19. Souyri, M., et al., Cell 63: 1137-1147 (1990).

20. Vigon, I., et al., Proc. Natl. Acad. Sci. USA 89: 5640-5644 (1992).

21. Skoda, R. C., et al., The EMBO Journal 12 (7): 2645-2653 (1993).

22. Ogawa, M., Blood 81 (11): 2844-2853 (1993).

23. Methia, N., et al., Blood 82 (5): 1395-1401 (1993).

24. Wendling, F, et al., Blood 80: 246a (1993).

D. The need for an agent capable of stimulating platelet production

It has been reported recently that platelet transfusions are beingadministered at an ever increasing rate at medical centers in NorthAmerica, Western Europe, and Japan. See Gordon, M. S. and Hoffman, R.,Blood 80 (2): 302-307 (1992). This increase appears to be due in largemeasure to advances in medical technology and greater access to suchtechnologies as cardiac surgery and bone marrow, heart, and livertransplantation. Dose intensification as a means of delivering therapiesto cancer patients and the HIV-1 epidemic have also contributed to theheavy demand on the platelet supply.

Platelet usage carries with it the possibility of transmission of themany blood-born infectious diseases as well as alloimmunization.Moreover, the production of purified platelets is an expensive endeavorand hence the increasing use of such platelets increases overall medicalcosts. As a result, there exists an acute need for new and improvedmethods for producing platelets for human uses.

Exemplary prior approaches to enhancing platelet production aredescribed in the following:

U.S. Pat. No. 5,032,396 reports that interleukin-7 (IL-7) is capable ofstimulating platelet production. Interleukin-7 is also known aslymphopoietin-1 and is a lymphopoietic growth factor capable ofstimulating growth of B- and T-cell progenitors in bone marrow.Published PCT application serial number 88/03747, filed Oct. 19, 1988and European patent application number 88309977.2, filed Oct. 24, 1988disclose DNA's, vectors, and related processes for producing mammalianIL-7 proteins by recombinant DNA technology. The data presented in theU.S. patent show that IL-7 can increase circulating platelets in normaland sublethally irradiated mice.

U.S. Pat. No. 5,087,448 discloses that megakaryocytes and platelets canbe stimulated to proliferate in mammals by treating them withinterleukin-6. Recombinant human interleukin-6 is a 26,000 molecularweight glycoprotein with multiple biological activities. The datapresented in this patent show that IL-6 has an effect of increasingcolonies of megakaryocytes in vitro.

None of the above-cited patents mentions anything with respect to theMpl ligands that are involved in the present invention.

In spite of the above disclosures, there remains a strong need for newstimulators of megakaryocytes and/or platelets in mammals.

E. Background relating to Chemically Modified MGDF

Proteins for therapeutic use are currently available in suitable formsin adequate quantities largely as a result of the advances inrecombinant DNA technologies. Chemical derivatives of such proteins mayeffectively block a proteolytic enzyme from physical contact with theprotein backbone itself, and thus prevent degradation. Additionaladvantages may include, under certain circumstances, increasing thestability and circulation time of the therapeutic protein and decreasingimmunogenicity. However, it should be noted that the effect ofmodification of a particular protein cannot be predicted. A reviewarticle describing protein modification and fusion proteins is Francis,Focus on Growth Factors 3: 4-10 (May 1992) (published by Mediscript,Mountview Court, Friern Barnet Lane, London N20, OLD, UK).

Polyethylene glycol ("PEG" or "peg") is one such chemical moiety whichhas been used in the preparation of therapeutic protein products. Forexample Adagen®, a formulation of pegylated adenosine deaminase isapproved for treating severe combined immunodeficiency disease;pegylated superoxide dismutase has been in clinical trials for treatinghead injury; pegylated alpha interferon has teen tested in phase Iclinical trials for treating hepatitis; pegylated glucocerebrosidase andpegylated hemoglobin are reported to have been in preclinical testing.For some proteins, the attachment of polyethylene glycol has been shownto protect against proteolysis, Sada, et al., J. FermentationBioengineering 71: 137-139 (1991), and methods for attachment of certainpolyethylene glycol moieties are available. See U.S. Pat. No. 4,179,337,Davis et al., "Non-Immunogenic Polypeptides," issued Dec. 18, 1979; andU.S. Pat. No. 4,002,531, Royer, "Modifying enzymes with PolyethyleneGlycol and Product Produced Thereby," issued Jan. 11, 1977. For areview, see Abuchowski et al., in Enzymes as Drugs. (J. S. Holcerbergand J. Roberts, eds. pp. 367-383 (1981)).

Other water soluble polymers have been used to modify proteins, such ascopolymers of ethylene glycol/propylene glycol, carboxymethylcellulose,dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, and poly aminoacids (either homopolymers or random copolymers).

For polyethylene glycol, a variety of means have been used to attach thepolyethylene glycol molecules to the protein. Generally, polyethyleneglycol molecules are connected to the protein via a reactive group foundon the protein. Amino groups, such as those on lysine residues or at theN-terminus, are convenient for such attachment. For example, Royer (U.S.Pat. No. 4,002,531, above) states that reductive alkylation was used forattachment of polyethylene glycol molecules to an enzyme. EP 0 539 167,published Apr. 28, 1993, Wright, "Peg Imidates and Protein DerivatesThereof" states that peptides and organic compounds with free aminogroup(s) are modified with an imidate derivative of PEG or relatedwater-soluble organic polymers. U.S. Pat. No. 4,904,584, Shaw, issuedFeb. 27, 1990, relates to the modification of the number of lysineresidues in proteins for the attachment of polyethylene glycol moleculesvia reactive amine groups.

One specific therapeutic protein which has been chemically modified isgranulocyte colony stimulating factor, "G-CSF." See European patentpublications EP 0 401 384, EP 0 473, 268, and EP 0 335 423.

Another example is pegylated IL-6, EP 0 442 724, entitled, "ModifiedhIL-6," (see co-pending U.S. Ser. No. 07/632,070) which disclosespolyethylene glycol molecules added to IL-6. EP 0 154 316, publishedSep. 11, 1985, reports reacting a lymphokine with an aldehyde ofpolyethylene glycol.

The ability to modify MGDF is unknown in the art since thesusceptibility of each individual protein to modification is determinedby the specific structural parameters of that protein. Moreover, theeffect of such a modification on the biological properties of eachprotein is unpredictable from the art. Because of the many clinicalapplications of MGDF, as set forth herein, a derivatized MGDF productwith altered properties is desirable. Such molecules may have increasedhalf-life and/or activity in vivo, as well other properties.

Pegylation of protein molecules will generally result in a mixture ofchemically modified protein molecules. As an illustration, proteinmolecules with five lysine residues and a free amino group at theN-terminus reacted in the above methods may result in a heterogeneousmixture, some having six polyethylene glycol moieties, some five, somefour, some three, some two, some one and some zero. And, among themolecules with several, the polyethylene glycol moieties may not beattached at the same location on different molecules. It will frequentlybe desirable to obtain a homogeneous product that contains substantiallyall one or a small number (e.g., 2-3) of modified protein species thatvary in the number and/or location of chemical moieties, such as PEG.Nevertheless, mixtures of, e.g., mono-, di- and/or tri-pegylated speciesmay be desirable or tolerable for a given therapeutic indication.

Variability of the mixture from lot to lot would be disadvantageous whendeveloping a therapeutic pegylated protein product. In such development,predictability of biological activity is important. For example, it hasbeen shown that in the case of nonselective conjugation of superoxidedismutase with polyethylene glycol, several fractions of the modifiedenzyme were completely inactive (P. McGoff et al. Chem. Pharm. Bull. 36:3079-3091 (1988)). See also, Rose et al., Bioconjugate Chemistry 2:154-159 (1991) which reports the selective attachment of the linkergroup carbohydrazide to the C-terminal carboxyl group of a proteinsubstrate (insulin). One cannot have such predictability if thetherapeutic protein differs in composition from lot to lot. Some of thepolyethylene glycol moieties may not be bound as stably in somelocations as others, and this may result in such moieties becomingdissociated from the protein. Of course, if such moieties are randomlyattached and therefore become randomly dissociated, the pharmacokineticsof the therapeutic protein cannot be precisely predictable.

Also highly desirable is a derivatized MGDF product wherein there is nolinking moiety between the polymer moiety and the MGDF moiety. Oneproblem with the above methods is that they typically require a linkingmoiety between the protein and the polyethylene glycol molecule. Theselinking moieties may be antigenic, which is also disadvantageous whendeveloping a therapeutic protein.

A method involving no linking group is described in Francis et al., In:"Stability of protein pharmaceuticals: in vivo pathways of degradationand strategies for protein stabilization" (Eds. Ahern., T. and Manning,M. C.) Plenum, N.Y., 1991) Also, Delgado et al. "Coupling of PEG toProtein By Activation with Tresyl Chloride, Applications InImmunoaffinity Cell Preparation", In: Fisher et al., ed., SeparationsUsing Aqueous Phase Systems, Applications In Cell Biology andBiotechnology, Plenum Press, N.Y., N.Y. 1989 pp. 211-213 involves theuse of tresyl chloride, which results in no linkage group between thepolyethylene glycol moiety and the protein moiety. This method may bedifficult to use to produce therapeutic products as the use of tresylchloride may produce toxic by-products.

Chamow et al., Bioconjugate Chem. 5: 133-140 (1994) report themodification of CD4 immunoadhesin with mono-methoxy-polyethylene glycol("MePEG glycol") aldehyde via reductive alkylation. The authors reportthat 50% of the CD4-Ig was mePEG-modified by selective reaction at theα-amino group of the N-terminus. Id. at page 137. The authors alsoreport that the in vitro binding capability of the modified CD4-Ig (tothe protein gp 120) decreased at a rate correlated to the extent ofMePEGylation. Ibid.

Thus, there is a need for MGDF derivatives, and, more particularly, aneed for pegylated MGDF. There also exists a need for methods to carryout such derivatization.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides novel polypeptides thatspecifically promote megakaryocyte growth and/or development ("Mplligands" or "MGDFs") which are substantially free from (i.e., isolatedfrom) other proteins (i.e., mammalian proteins in the case of an Mplligand obtained from a mammalian source). Such proteins may be purifiedfrom cell sources producing the factors naturally or upon induction withother factors. They may also be produced by recombinant geneticengineering techniques. Mpl ligands may further be synthesized bychemical techniques, or a combination of the above-listed techniques.

The Mpl ligands of this invention are obtainable in their native formfrom mammalian sources. Two exemplary Mpl ligands isolated from canineaplastic plasma are described in the examples section herein. However,it is demonstrated in other examples herein that closely related Mplligands are present in aplastic plasma from both human and porcinesources. Notably, the activity of each of the human, porcine, and canineMpl ligands is specifically inhibitable by the soluble form of themurine Mpl receptor, demonstrating that all of these Mpl ligands (aswell as those from other mammalian sources, including murine) areclosely related both on structural and activity levels.

It is expected that human, porcine, and other mammalian Mpl ligands, maybe isolated from natural sources by procedures substantially as detailedherein. See Example 10. Accordingly, this invention generallyencompasses mammalian Mpl ligands, such as from dogs, pigs, humans,mice, horses, sheep, and rabbits. Particularly preferred Mpl ligands arethose from dogs, pigs and humans.

In addition, genes encoding human Mpl ligands have been cloned from ahuman fetal kidney and liver libraries and sequenced, as set forth inthe Example section below. Two human polypeptide sequences have beendetermined to have activity in a cell-based assay (see Example 4). Thesesequences differ in their length, but have identity over a large stretchof their amino acid sequences. The identical portions have homology toerythropoietin. The Mpl ligands are also referred to herein asMegakaryocyte Growth and Development Factors (MGDFs); all generalreferences to Mpl ligands shall apply to those referred to herein asMGDFs and vice versa. By "MGDF polypeptide" is meant a polypeptide thathas an activity to specifically stimulate or inhibit the growth and/ordevelopment of megakaryocytes. Exemplary such polypeptides are disclosedherein.

The Mpl ligands of the present invention have been found to bespecifically active in the megakaryocyte lineage, augmenting maturationand/or proliferation of megakaryocytes, as demonstrated in the assays ofExamples 2 and 4 below. By "specifically" is meant that the polypeptidesexhibit biological activity to a relatively greater degree towardsmegakaryocytes as compared to many other cell types. Those that arestimulatory towards megakaryocytes are expected to have an in vivoactivity of stimulating the production of platelets, through thestimulation of maturation and differentiation of megakaryocytes.

Two preferred Mpl ligands from a canine source have apparent molecularweights of approximately 25 kd and 31 kd as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducingconditions. Both proteins are purified during the same purificationprotocol which is detailed in the examples section below.

Two preferred human ligands, MGDF-1 (amino acids 1-332 of SEQ ID NO: 25)and MGDF-2, (amino acids 1-174 of SEQ ID NO: 25) are 332 and 174 aminoacids in length, respectively, not including a 21 amino acid putativesignal peptide. These sequences, and a third related molecule, MGDF-3(amino acids 1-265 of SEQ ID NO: 27) are shown in FIGS. 11 and 12.

Still a further aspect of the present invention are processes forisolating and purifying the Mpl ligands of the present invention orfragments thereof from mammalian sources, preferably whole blood, serumor plasma. Aplastic blood, serum or plasma are especially preferredstarting materials. Aplastic blood, serum or plasma may be obtained by aprocess involving irradiating a mammal with a radiation source such ascobalt-60 at a radiation level of about 400-800 rads so as to renderthem aplastic. Such a procedure is known in the art, as exemplified inthe publications cited in Example 1 below. In the case of humans,irradiated blood, plasma, or serum may be obtained from a patient afterradiation therapy, e.g., to treat cancer.

Thereafter, the aplastic blood, serum or plasma is subjected to apurification process. The purification process provided by the presentinvention comprises the following key procedures: lectin affinitychromatography and Mpl receptor affinity chromatography. Each of theseprocedures results in an approximately 300-500-fold purification of the25 and 31 kd proteins from canine aplastic plasma. Other standardprotein purification procedures may be included with the aboveprocedures to further purify the Mpl ligands of the present invention,such as those procedures detailed below.

Another aspect of the present invention includes polynucleotides thatencode the expression of a mammalian Mpl ligand protein. Such DNAsequences may include an isolated DNA sequence that encodes theexpression of mammalian Mpl ligand proteins as described herein. The DNAsequences may also include 5' and 3' mammalian non-coding sequencesflanking the Mpl ligand coding sequence. The DNA sequences may furtherencode an amino terminal signal peptide.

Also provided by the present invention are recombinant DNA molecules,each comprising vector DNA and a DNA sequence encoding a mammalian Mplligand. The DNA molecules provide the Mpl ligand DNA in operativeassociation with a regulatory sequence capable of directing thereplication and expression of Mpl ligand in a selected host cell. Hostcells (e.g., bacterial, mammalian insect, yeast, or plant cells)transformed with such DNA molecules for use in expressing a recombinantMpl ligand protein are also provided by the present invention.

The DNA molecules and transformed cells of the invention are employed inanother aspect, a novel process for producing recombinant mammalian Mplligand protein, or peptide fragments thereof. In this process a cellline transformed with a DNA sequence encoding expression of Mpl ligandprotein or a fragment thereof (or a recombinant DNA molecule asdescribed above) in operative association with a suitable regulatory orexpression control sequence capable of controlling expression of theprotein is cultured under appropriate conditions permitting expressionof the recombinant DNA. This claimed process may employ a number ofknown cells as host cells for expression of the protein. Presentlypreferred cell lines for producing Mpl ligand are mammalian cell lines(e.g., CHO cells) and bacterial cells (e.g., E. coli).

The expressed Mpl ligand protein is then harvested from the host cell,cell lysate or culture medium by suitable conventional means. Theconditioned medium may be processed through the same purification stepsor modifications thereof as used to isolate the Mpl ligand from aplasticplasma. (See Example 7).

In a still further aspect of the present invention, there are providedrecombinant Mpl ligand proteins. These proteins are substantially freefrom other mammalian materials, especially proteins. The Mpl ligandproteins of this invention are also characterized by containing one ormore of the physical, biochemical, pharmacological or biologicalactivities described herein.

The present invention also relates to chemically modified MGDF comprisedof a MGDF protein moiety connected to at least one water solublepolymer, and methods for the preparation and use of such compositions.In particular, the present invention includes chemically modified MGDFwherein the MGDF species is reacted with reactive polyethylene glycolmolecules so as to attach PEG to MGDF. Such attachment may beaccomplished by pegylation reactions discussed herein, such as acylationor alkylation. Acylation or alkylation with PEG may be carried out underconditions whereby the major product is monopegylated or polypegylated.Polypegylation generally involves attachment of PEG to the ε-aminogroups of lysine residues and may additionally involve pegylation at theN-terminus of the polypeptide. Monopegylation preferably involvesattachment of PEG to the α-amino group at the N-terminus of the protein.The yield and homogeneity of such monopegylation reaction may beenhanced via a type of reductive alkylation which selectively modifiesthe α-amino group of the N-terminal residue of an MGDF protein moiety,thereby providing for selective attachment of a water soluble polymermoiety at the N-terminus of the protein. This provides for asubstantially homogeneous preparation of polymer/MGDF protein conjugatemolecules as well as (if polyethylene glycol is used) a preparation ofpegylated MGDF protein molecules having the polyethylene glycol moietydirectly coupled to the protein moiety.

Another aspect of this invention provides pharmaceutical compositionscontaining a therapeutically effective amount of isolatednaturally-occurring or recombinant Mpl ligand, which may be derivatizedwith a water soluble polymer such as polyethylene glycol, along with apharmaceutically acceptable carrier, diluent, or excipient. Thesepharmaceutical compositions may be employed in methods for treatingdisease states or disorders characterized by a deficiency ofmegakaryocytes and/or platelets as well as an in vivo deficiency of theMpl ligand. They may also be employed prophylactically to ameliorateexpected megakaryocyte or platelet deficiencies (e.g., due to surgery).

Thus, the Mpl ligands of the present invention may be employed in thetreatment of aplastic anemias, e.g., to augment production of plateletsin patients having impaired platelet production (such as AIDS patientsor patients undergoing cancer chemotherapy). Mpl ligand may be used totreat blood disorders such as thrombocytopenia. Mpl ligand may be usedas an adjunctive therapy for bone marrow transplant patients. Suchpatients could be human or another mammal. Mpl ligand from one speciesis also expected to be useful in another species.

A further aspect of the invention, therefore, is a method for treatingthese and other pathological states resulting from a deficiency ofplatelets by administering to a patient a therapeutically effectiveamount of a pharmaceutical composition as described above. Thesetherapeutic methods may include administration, simultaneously orsequentially with Mpl ligand, an effective amount of at least one othermegakaryocyte colony stimulating factor, a cytokine (e.g., EPO), asoluble Mpl receptor, hematopoietin, interleukin, growth factor, orantibody.

Still another aspect of the present invention provides antibodies (e.g.,polyclonal, monoclonal, humanized, and recombinant), and antibodyfragments, directed against (i.e., reactive with) a mammalian Mpl ligandor a ligand fragment. As part of this aspect, therefore, the inventionincludes cells capable of secreting such antibodies (e.g., hybridomas inthe case of monoclonal antibodies) and methods for their production anduse in diagnostic or therapeutic procedures.

Another aspect of the invention is an assay of a body fluid for thepresence of Mpl ligand. Such an assay could employ antibodies thatspecifically recognize an Mpl ligand, in a single antibody or "sandwich"format. Such an assay could be used to determine whether a patient needsexternal administration of Mpl ligand and/or whether such patient islikely to experience a platelet deficiency or disorder. Such assayscould be included in a kit format, including positive and negativecontrols, antibody(ies), and other standard kit components.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description of preferredembodiments thereof.

BRIEF DESCRIPTION OF THE FIGURES

Numerous features and advantages of the present invention will becomeapparent upon review of the figures, wherein:

FIG. 1 depicts an overview of development and maturation ofmegakaryocytes and platelets.

FIG. 2 demonstrates that soluble murine Mpl receptor substantiallycompletely inhibits the ability of plasma from irradiated dogs("aplastic canine" or "APK9"), to induce megakaryocyte development. Theassay for megakaryocyte development was that described in Example 2.

FIG. 3 shows that an activity enriched from APK9 by lectin affinity andMpl receptor affinity chromatography procedures ("Mpl ligand")stimulates the growth of 1A6.1 cells and that soluble murine Mplreceptor blocks that growth.

FIG. 4 shows an overview of the purification steps involved in purifyingthe 25 and 31 kd forms of the canine Mpl receptor from aplastic canineplasma.

FIG. 5A shows the purification of Mpl ligand by reversed phase HPLC(RP-HPLC).

FIG. 5B shows that fraction 21 from the RP-HPLC contains highly purified31 kd Mpl ligand; fraction 22 contains a mixture of the 31 kd and 25 kdMpl ligands; and fraction 23 contains highly purified 25 kd Mpl ligand.

FIG. 6 shows a comparison of Mpl ligand activities in reverse phase HPLC(C4 column) fractions that contain the 25 and/or 31 kd Mpl ligandproteins.

FIG. 7 shows the number of megakaryocytes produced from cultures ofCD34-selected peripheral blood cells stimulated with APK9, Mpl ligandand various other factors.

FIG. 8 shows the number of total leukocytes produced from cultures ofCD34-selected peripheral blood cells stimulated with APK9, Mpl ligandand various other factors.

FIG. 9 shows the percentages of megakaryocytes that are produced incultures of CD34-selected peripheral blood cells stimulated with APK9,Mpl ligand and various other factors.

FIG. 10 shows that human IL-3 is not involved in Mpl ligand-inducedmegakaryocyte development.

FIG. 11 shows the cDNA and deduced amino acid sequences of human MGDF-1(amino acids 1-332 of SEQ ID NO: 25) and MGDF-2 (amino acids 1-174 ofSEQ ID NO: 25).

FIG. 12 shows the cDNA and deduced amino acid sequences of human MGDF-3(amino acids 1-265 of SEQ ID NO: 27).

FIG. 13A shows a comparison between MGDF- 1 (amino acids 1-332 of SEQ IDNO: 25) with a signal peptide attached to the N-terminus and MGDF from acanine source also having a signal peptide attached to the N-terminus.

FIG. 13B shows a comparison between MGDF-1 (amino acids 1-332 of SEQ IDNO: 25) and MGDF with a signal peptide attached to the N-terminus from amurine source also having a signal peptide attached to the N-terminus.

FIG. 14 shows an example of MGDF acylation using N-hydroxysuccinimidyl(NHS) active esters of mono-methoxy-polyethylene glycol to result in apoly-pegylated product.

FIG. 15 shows an example of nonspecific MGDF reductive alkylation usingmono-methoxy-polyethylene glycol aldehydes to result in a poly-pegylatedproduct.

FIG. 16 shows an example of site-specific MGDF reductive alkylation atthe α-amino group of the N-terminal residue usingmono-methoxy-polyethylene glycol aldehydes to result in a substantiallymono-pegylated product.

FIG. 17A shows size exclusion (SEC) HPLC analysis of poly-MePEG-MGDFconjugate prepared by MGDF acylation with the NHS ester of 20 kDa MePEG(PEG 11).

FIG. 17B shows SEC HPLC analysis of poly-MePEG-MGDF conjugate preparedby MGDF alkylation with 20 kDa MePEG aldehyde (PEG 20).

FIG. 17C shows SEC HPLC analysis of mono-MePEG-MGDF conjugate preparedby MGDF alkylation with MW 20 kDa MePEG aldehyde (PEG 16).

FIG. 18 shows platelet counts from mice treated with recombinant humanMGDF: open diamond=CHO-derived 1-332 MGDF (MGDF-1; amino acids 1-332 ofSEQ ID NO: 25) open circles=unpegylated E. coli 1-163 MGDF (MGDF-11;amino acids 1-163 of SEQ ID NO: 25) and closed circles=pegylated E. coli1-163 MGDF (MGDF-11; amino acids 1-163 of SEQ ID NO: 25).

DETAILED DESCRIPTION OF THE INVENTION

Additional aspects and advantages of the invention will be apparent tothose skilled in the art upon consideration of the followingdescription, which details the practice of the invention.

The novel mammalian megakaryocyte growth promoting, and/or plateletproducing factors, referred to as Mpl ligands, provided by the presentinvention are homogeneous proteins substantially free of associationwith other mammalian proteinaceous materials. Preferably, the proteinsare about 90% free of other proteins, particularly preferably, about 95%free of other proteins, and most preferably about ≧98% free of otherproteins. These proteins can be produced via recombinant techniques toenable large quantity production of pure, active Mpl ligand useful fortherapeutic applications. Alternatively such proteins may be obtained ina homogeneous form from mammalian aplastic blood, plasma or serum, orfrom a mammalian cell line secreting or expressing an Mpl ligand.Further, Mpl ligand or active fragments thereof may be chemicallysynthesized.

In general, by "Mpl ligands" as used in connection with the presentinvention is meant the Mpl ligands disclosed herein as well as activefragments and variants thereof, which are described in greater detailbelow.

Two preferred Mpl ligands from a canine source have apparent molecularweights of approximately 25 kd and 31 kd as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducingconditions. Both proteins are purified during the same purificationprotocol which is detailed in the examples section below. Thus, forexample, both of these Mpl ligands bind wheat germ lectin andimmobilized Mpl receptor. The 25 kd form includes an amino acid sequenceas follows:

Ala-Pro-Pro-Ala-Xaa-Asp-Pro-Arg-Leu-Leu-Asn-Lys-Met-Leu-Arg-Asp-Ser-His-Val-Leu-His-Xaa-Arg-Leu-Xaa-Gln-Xaa-Pro-Asp-Ile-Tyr(SEQ ID NO: 1).

The 31 kd form includes an amino acid sequence as follows:

Ala-Pro-Pro-Ala-Xaa-Asp-Pro-Arg-Leu-Leu-Asn-Lys-Met-Leu-Arg-Asp-Ser-His-Val-Leu-His(SEQ ID NO: 2).

The "Xaa" amino acids shown in SEQ ID NOS: 1 and 2 are not known withcertainty, but are expected to be cysteine, serine, threonine, or (lesslikely) tryptophan.

It can be seen from the above sequences that the 31 kd ligand comprisesat least a portion of the 25 kd form. In particular, the first 21 aminoacids of the 31 kd protein are exactly the same as those of the 25 kdprotein. This evidence, and especially the fact that both proteins haveactivity in the Mpl ligand activity assays presented herein, leads tothe conclusion that both proteins are very closely related in terms ofstructure and activity. It is likely that the 31 kd form of the proteindiffers form the 25 kd form in differential C-terminal sequence,differential glycosylation and/or differential splicing of the geneencoding the proteins.

In addition to the above sequence information, another sequence wasdetermined during sequencing of the 25 kd band prior to the finalpurification step (using reverse phase HPLC). This sequence was foundassociated with the 25 kd band under non-reducing conditions but notreducing conditions, implying that it is the result of cleavage into twoportions (e.g., by a protease) of the 25 kd protein, which portions areheld together by a disulfide bond. This sequence is:

Thr-Gln-Lys-Glu-Gln-Thr-Lys-Ala-Gln-Asp-Val-Leu-Gly-Ala-Val-Ala-Leu (SEQID NO: 3)

Although the precise location of SEQ ID NO: 3 in the sequence of the 25kd protein is unclear, analogy with other cytokines, such aserythropoietin, supports the possibility that the sequence occurs aroundamino acid number 114 in the 25 kd protein. It should be noted that itis likely, although unproven, that SEQ ID NO: 3 also occurs in the 31 kdprotein, probably again starting around amino acid number 114. Thissequence information is discussed in additional detail in Example 7.

Since the initial purification experiments of the canine ligands,summarized above, a gene encoding a canine ligand has now been cloned.As a result, the full length amino acid sequence of this canine ligandhas been determined to be that set forth in FIG. 13A. Based on molecularweight calculations, it is predicted that the 25 kd and 31 kd canineligands are C-terminal processed forms of the full-length ligand shownin FIG. 13A. Additionally, a murine Mpl ligand has been obtained havingthe sequence set forth in FIG. 3B.

Such purified ligands may also be characterized by specific activity inthe human megakaryocyte assay of Example 2 of at least about 5.7×10⁹megakaryocyte units/mg. A megakaryocyte unit is defined as that amountof material that results in the production of as many megakaryocytes as1 microliter of APK9 standard control using the assay described inExample 2.

Such purified ligands are also characterized by a specific activity inthe Mpl-dependent cell growth assay of Example 4 of at least about6.9×10⁹ cell growth units/mg. A "cell growth unit" is defined as theamount of ligand required to result in the growth of 200 1A6.1 cells inthe assay of Example 4.

The following Table 1 shows additional specific calculations of activityfor actual purified canine Mpl ligands prepared in accordance with thisinvention:

                  TABLE 1                                                         ______________________________________                                        Mp1         1A6.1 assay                                                                             Human Meg assay                                         Ligand      (units/mg)                                                                              (units/mg)                                              ______________________________________                                        31 kd       6.52 × 10.sup.9                                                                   5.7 × 10.sup.9                                    25 kd       10.5 × 10.sup.9                                                                    14 × 10.sup.9                                    ______________________________________                                    

Summarizing the above information, some exemplary Mpl ligands of thepresent invention are characterized by one or more of the followingbiochemical and biological properties:

(a) such Mpl ligands are isolated from canine aplastic plasma;

(b) such Mpl ligands have apparent molecular weights of approximately 25kd or 31 kd as determined by 12-14% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under non-reducingconditions;

(c) the Mpl ligands comprise the following amino acid sequences:

SEQ ID NO: 1, in the case of the 25 kd protein; or

SEQ ID NO: 2, in the case of the 31 kd protein;

(d) the Mpl ligands additionally comprise the amino acid sequence SEQ IDNO: 3 (particularly preferably in the 25 kd protein);

(e) the Mpl ligands bind to wheat germ lectin;

(f) the Mpl ligands bind to immobilized soluble murine Mpl receptor;

(g) the Mpl ligand activity can be inhibited in vitro by soluble Mplreceptor; and

(h) the Mpl ligands bind to an anion exchange column at a pH of about8-9.

The biological activities of preferred Mpl ligands of the presentinvention are demonstrated by their ability to specifically stimulatethe growth and development of megakaryocytes in the megakaryocyte growthpromoting assay of Example 2. In this assay, MPL ligand stimulates thedifferentiation of human peripheral blood CD34⁺ (i.e., CD-34 cellsselected by immunoadsorption) cells during an 8 day culture period.Megakaryocytes are identified by staining with specific anti-plateletantigen antibodies and counted microscopically. MPL ligand alsostimulates the growth of the factor-dependent cell line, 1A6.1. In theabsence of MPL ligand, the cells will die. 1A6.1 cell number is assessedafter 2 days in culture with MPL ligand.

The Mpl ligands described above have specific activities as described inTable 1 above.

Sources of the Mpl ligands have been determined to be aplastic mammalianblood, plasma, or serum. However, the source of such ligands is notexpected to be limited to such known sources and may include othermammalian body fluids, cells obtained therefrom, etc.

The purification of native Mpl ligands from mammalian sources is basedon two key purification steps:

(a) lectin affinity chromatography, preferably using wheat germagglutinin; and

(b) immobilized Mpl receptor affinity chromatography.

Additional steps may be included to further purify the protein, such asion exchange chromatography, gel filtration chromatography, and reversephase chromatography.

The purification techniques actually employed in obtaining Mpl ligandfrom canine aplastic plasma comprise the following steps (See, Example7).

(a) lectin affinity chromatography (wheat germ agglutinin chromatographyis especially preferred);

(b) soluble Mpl receptor (Mpl-X) affinity chromatography (preferably,using immobilized murine Mpl-X);

(c) ion (anion or cation) exchange chromatography (preferably, anionexchange chromatography; particularly preferably using a Mono Q column);

(d) gel filtration chromatography under dissociative conditions(preferably, using Superdex 200 plus SDS); and

(e) reverse phase chromatography (preferably, using a C-4 column).

Homogeneous mammalian Mpl ligand, including the human ligand, may beobtained by applying the above purification procedures to aplasticblood, serum, or plasma or other sources of mammalian Mpl ligand, e.g.,cell or tissue sources. The steps are not required to be in a particularsequence, but the listed sequence is preferred. Procedures for culturinga cell (or tissue) source which may be found to produce Mpl ligand areknown to those of skill in the art and may be used, for example, toexpand the supply of starting material.

Mpl ligand or one or more peptide fragments thereof may also be producedvia recombinant techniques. To obtain the DNA sequence for a particularMpl ligand, the purified Mpl ligand material is reduced and optionallydigested with a protease such as trypsin. Enzymatic fragments areisolated and sequenced by conventional techniques. Alternatively, asexemplified in the examples herein, the intact purified protein may besequenced directly to the extent possible based on the quantity ofprotein available and then the sequenced region may be used analogouslyto the sequenced tryptic fragments in the following procedure.Oligonucleotide probes are synthesized using the genetic code to predictall possible sequences that encode the amino acid sequences of thesequenced fragment(s). Preferably, several degenerate sequences aregenerated as probes. The Mpl ligand gene is identified by using theseprobes to screen a mammalian genomic library or other source.Alternatively, the mRNA from a cell source of Mpl ligand can be used tomake a cDNA library which can be screened with the probes to identifythe cDNA encoding the Mpl ligand polypeptide. Further, the PCR techniquemay be used to extend the cDNA sequence, using appropriate primers.

Using these probes to screen a genomic library, a DNA clone is obtained.To obtain a full length clone, probes based on the obtained DNA sequencemay be employed to rescreen the library and hybridize to the full lengthMpl ligand DNA sequence.

The human cDNA for Mpl ligand can also be obtained by subcloning a fulllength human genomic clone into an expression vector, transfecting itinto COS cells, preparing a cDNA library from these transfected COScells and screening by hybridization for Mpl ligand cDNA. Once theentire cDNA is identified, it or any portion of it that encodes anactive fragment of Mpl ligand, can be introduced into any one of avariety of expression vectors to make an expression system for Mplligand or one or more fragments thereof.

By such use of recombinant techniques, preferred DNA sequences encodingthe Mpl ligand polypeptide are obtained. The present invention alsoencompasses these DNA sequences, free of association with DNA sequencesencoding other proteins (i.e., isolated), and coding for expression ofMpl ligand polypeptides with an Mpl ligand activity (e.g., megakaryocytegrowth and/or development). These DNA sequences include those sequencesencoding all or a fragment of Mpl ligand and those sequences whichhybridize, preferably under stringent hybridization conditions to thecDNA sequences See, T. Maniatis et. al., Molecular Cloning (A LaboratoryManual); Cold Spring Harbor Laboratory (1982), pages 387-389!.

Exemplary stringent hybridization conditions are hybridization in 4×SSCat 62°-67° C., followed by washing in 0.1× SSC at 62°-67° C. forapproximately an hour. Alternatively, exemplary stringent hybridizationconditions are hybridization in 45-55% formamide, 4×SSC at 40°-45° C.

DNA sequences which hybridize to the sequences for Mpl ligand underrelaxed hybridization conditions and which encode Mpl ligand peptideshaving Mpl ligand biological properties also encode novel Mpl ligandpolypeptides of this invention. Examples of such relaxed stringencyhybridization conditions are 4×SSC at 45°-55° C. or hybridization with30-40% formamide at 40°-45° C. For example, a DNA sequence which sharesregions of significant homology, e.g., sites of glycosylation ordisulfide linkages, with the sequences of Mpl ligand and encodes aprotein having one or more Mpl ligand biological properties clearlyencodes an Mpl ligand polypeptide even if such a DNA sequence would notstringently hybridize to the Mpl ligand sequence(s).

Allelic variations (naturally-occurring base changes in the speciespopulation which may or may not result in an amino acid change) of DNAsequences encoding the peptide sequences of Mpl ligand are also includedin the present invention, as well as analogs or derivatives thereof.Similarly, DNA sequences which code for Mpl ligand polypeptides butwhich differ in codon usage due to the degeneracies of the genetic codeor variations in the DNA sequence of Mpl ligand which are caused bypoint mutations or by induced modifications to enhance the activity,half-life or production of the polypeptides encoded thereby are alsoencompassed in the invention.

A cloning procedure as set forth in Example 11 below was followed andresulted in the amino acid and cDNA sequences of the human proteinsMGDF-1 (amino acids 1-332 of SEQ ID NO: 25), MGDF-2 (amino acids 1-174of SEQ ID NO: 25), and MGDF-3 (amino acids 1-265 of SEQ ID NO: 27)disclosed herein. MGDF-1 (amino acids 1-332 of SEQ ID NO: 25) is shownas amino acids 1-332 in FIG. 11 and contains 332 amino acids. MGDF-2(amino acids 1-174 of SEQ ID NO: 25) is a truncated portion ofMGDF-1(amino acids 1-332 of SEQ ID NO: 25), and is shown as amino acids1-174 in FIG. 11. MGDF-2 (amino acids 1-174 of SEQ ID NO: 25) thereforecontains 174 amino acids. MGDF-3 (amino acids 1-265 of SEQ ID NO: 27) isshown as amino acids 1-265 in FIG. 12 and contains 265 amino acids. Ineach MGDF disclosed herein, the molecule including the signal peptide,shown as amino acids -21 to -1 in both FIGS. 11 and 12, is also part ofthe present inventive polypeptides, but it is preferably removed formegakaryocyte growth and development activity to be exhibited. Insummary, MGDFs 1-3 are defined as follows:

MGDF-1 amino acids 1-332 SEQ ID NO: 25

MGDF-2 amino acids 1-174 SEQ ID NO: 25

MGDF-3 amino acids 1-265 SEQ ID NO: 27.

Based on the activity data presented herein, it is hypothesized thathuman MGDF is expressed in vivo as a substantially inactive or lessactive precursor polypeptide that contains variable C-terminal aminoacids. Upon cleavage of the C-terminal amino acids (as well as thesignal peptide), the processed form(s) of the molecule retain activityor become more active.

In the assays presented herein, MGDF-1 (amino acids 1-332 of SEQ ID NO:25) and MGDF-2 (amino acids 1-174 of SEQ ID NO: 25) were active whereasMGDF-3 (amino acids 1-265 of SEQ ID NO: 27) was not. In view of theabove hypothesis, it is believed that MGDF-1 (amino acids 1-332 of SEQID NO: 25) may require processing (e.g., cleavage with a protease) inorder to exhibit its activity. The fact that a truncated form of MGDF-1(amino acids 1-332 of SEQ ID NO: 25) (i.e., MGDF-2(amino acids 1-174 ofSEQ ID NO: 25)) is active supports this hypothesis.

Conditioned medium from human kidney 293 cells (Invitrogen) transfectedwith the MGDF-1 gene (nucleotides 99-1094 of SEQ ID NO: 24) doesdemonstrate activity in the cell assay of Example 4 below. On the otherhand, in other cell lines, e.g., 32 D cells, no activity was seen forthis molecule. It is hypothesized that this may mean that 293 cells areable to process the MGDF-1 (amino acids 1-332 of SEQ ID NO: 25)molecule, presumably by truncation, so that the molecule primarilyresponsible for the activity is a truncated form, whereas the 32 D cellsare unable to process the molecule.

In view of the above hypothesis, various active molecules may resultfrom truncations of the sequence set forth as MGDF-1 (amino acids 1-332of SEQ ID NO; 25) (FIG. 11). Structural features conserved among thecytokine family, such as erythropoietin (EPO), include four α-helicalbundles and four cysteines. Referring to the MGDF-1 (amino acids 1-332of SEQ ID NO: 25) sequence, Cys 151 is the most C-terminal element ofthese evolutionarily conserved and functionally essential structures.Therefore, preferred truncation variants of MGDF-1 (amino acids 1-332 ofSEQ ID NO: 25) are any of those that have C-terminal truncations fromamino acid 152 to 332 (along with cleavage of the signal peptide).Preferably, the sequence of MGDF-1 (amino acids 1-332 of SEQ ID NO: 25)will have removed from it from 50 to 185 amino acids from theC-terminus, particularly preferably, from 90 to 172 amino acids from theC terminal. As disclosed herein, the signal peptide is thought to be 21amino acids in length; however, the signal peptide may have 23 aminoacids, based on the sequence of MGDF-1(amino acids 1-332 of SEQ ID NO:25). Accordingly, polypeptides corresponding to those presented hereinbut which start at position 3 of FIG. 11 or 12 are also specificallycontemplated.

The following are some specific preferred variants of MGDF-1 (aminoacids 1-332 of SEQ ID NO: 25) that may exhibit activity (i.e., theability to promote the growth of megakaryocytes and/or platelets; orinhibitory/ stimulatory activity towards the natural receptor):

MGDF-4 amino acids 1-151 SEQ ID NO: 25

MGDF-5 amino acids 1-156 SEQ ID NO: 25

MGDF-6 amino acids 1-170 SEQ ID NO: 25

MGDF-7 amino acids 1-177 SEQ ID NO: 25

MGDF-8 amino acids 1-244 SEQ ID NO: 25.

In some clones, amino acids 112-115 in the MGDF-1 (amino acids 1-332 ofSEQ ID NO: 25) sequence were absent, so that sequences corresponding tothose set forth above, but in which these amino acids are missing (andthe C-terminus amino acid number adjusted down by 4) may also be active.

In one clone, which had a termination codon at position 171, an Alaresidue was found instead of a Thr residue as shown at position 170 inFIG. 11. Therefore, the invention includes variants of MGDF molecules inwhich position 170 is Ala instead of Thr.

MGDF-3 (amino acids 1-265 of SEQ ID NO: 27) results from removal of asequence referred to herein as IVS-5 (Intervening Sequence-5) since thissequence is spliced within the fifth exon in the sequence. Since the 5'end of IVS-5 occurs within a codon, its removal results in a frame-shiftin the remaining sequence of MGDF, which can be seen to occur startingat position 139 of MGDF-3 (amino acids 1-265 of SEQ ID NO: 27) to theend of the molecule.

No activity has yet been found for MGDF-3 (amino acids 1-265 of SEQ IDNO: 27) itself upon transfection into 293 cells and testing theresulting conditioned medium for activity in the cell-based assay ofExample 4. Apparently, unlike MGDF-1 (amino acids 1-332 of SEQ ID NO:25), 293 cells are unable to process MGDF-3 (amino acids 1-265 of SEQ IDNO: 27) to an active form. Nevertheless, based on the truncationhypothesis set forth above in connection with MGDF-1 (amino acids 1-332of SEQ ID NO: 25), truncation of C-terminal amino acids from MGDF-3(amino acids 1-265 of SEQ ID NO: 27) may also result in activity. Forexample, C-terminal truncation of MGDF-3 (amino acids 1-265 of SEQ IDNO: 27) of from 40 to 102 amino acids may result in activity.Preferably, from 50 to 90 amino acids are removed. Two specificpreferred variants of MGDF-2 (amino acids 1-174 of SEQ ID NO: 25) are:

MGDF-9 amino acids 1-158 of SEQ ID NO: 27

MGDF-10 amino acids 1-244 of SEQ ID NO: 27

In all of the Mpl ligands disclosed herein, including the exemplaryMGDFs set forth above, a methionyl residue may be present at theN-terminus, especially when such polypeptides are expressed in bacterialhost cells.

Mpl ligand polypeptides may also be produced by known conventionalchemical synthesis. Methods for constructing the polypeptides of thepresent invention by synthetic means are known to those of skill in theart. The synthetically-constructed Mpl ligand polypeptide sequences, byvirtue of sharing primary, secondary, or tertiary structural andconformational characteristics with Mpl ligand polypeptides may possessMpl ligand biological properties in common therewith. Thus, they may beemployed as biologically active or immunological substitutes fornatural, purified Mpl ligand polypeptides in therapeutic andimmunological processes.

Modifications in the peptides or DNA sequences encoding Mpl ligand canbe made by one skilled in the art using known techniques. Modificationsof interest in the Mpl ligand sequences may include the replacement,insertion or deletion of a selected amino acid residue in the codingsequences. Mutagenesis techniques for such replacement, insertion ordeletion are well known to one skilled in the art. See, e.g., U.S. Pat.No. 4,518,584.! Conservative changes in from 1 to 20 amino acids arepreferred. Preferred peptides may be generated by proteolytic enzymes,or by direct chemical synthesis. Such variants are included within themeaning of Mpl ligand polypeptides and polynucleotides of the presentinvention.

Specific mutations of the sequences of the Mpl ligand polypeptide mayinvolve modifications of a glycosylation site (e.g., serine, threonine,or asparagine). The absence of glycosylation or only partialglycosylation results from amino acid substitution or deletion at anyasparagine-linked glycosylation recognition site or at any site of themolecule that is modified by addition of O-linked carbohydrate. Anasparagine-linked glycosylation recognition site comprises a tripeptidesequence which is specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences are either Asn-Xaa-Thror Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro. Avariety of amino acid substitutions or deletions at one or both of thefirst or third amino acid positions of a glycosylation recognition site(and/or amino acid deletion at the second position) results innon-glycosylation at the modified tripeptide sequence. Expression ofsuch altered nucleotide sequences produces variants which are notglycosylated at that site.

Additional Analogs/Derivatives of MGDF

Other analogs and derivatives of the sequences of MGDF (Mpl ligands),which may retain MGDF (Mpl ligand) activity in whole or in part may alsobe prepared by one of skill in the art given the disclosures herein.Such modifications are also encompassed by this invention.

More particularly, the present invention also broadly includeschemically modified MGDF compositions and methods of making and usingthem. The present disclosure reveals that it is possible to modify MGDFand to enhance its properties.

In one aspect, the present invention relates to an MGDF productcomprising an MGDF protein linked to at least one water soluble polymermoiety.

In another aspect, the present invention relates to an MGDF productwherein said MGDF protein is linked to at least one polyethylene glycolmolecule.

In another aspect, the present invention relates to MGDF moleculesattached to at least one polyethylene glycol molecule via an acyl oralkyl linkage.

Pegylation of MGDF may be carried out by any of the pegylation reactionsknown in the art. See, for example: Focus on Growth Factors 3 (2): 4-10(1992); EP 0 154 316; EP 0 401 384; and the other publications citedherein that relate to pegylation. Preferably, the pegylation is carriedout via an acylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule (or an analogous reactive water-solublepolymer). These preferred means for derivatization with polyethyleneglycol will now be discussed in greater detail.

Acylation

Pegylation by acylation generally involves reacting an active esterderivative of polyethylene glycol (PEG) with an MGDF protein. Any knownor subsequently discovered reactive PEG molecule may be used to carryout the pegylation of MGDF. A preferred activated PEG ester is PEGesterified to N-hydroxysuccinimide ("NHS"). As used herein, "acylation"is contemplated to included without limitation the following types oflinkages between MGDF and a water soluble polymer such as PEG: amide,carbamate, urethane, and the like. See Bioconjugate Chem. 5: 133-140(1994). Reaction conditions may be selected from any of those known inthe pegylation art or those subsequently developed, but should avoidconditions such as temperature, solvent, and pH that would inactivatethe MGDF species to be modified. Reaction conditions that applygenerally to pegylation of MGDFs will be described below. An exemplaryreaction with an NHS ester of monomethoxy-PEG is depicted in FIG. 14.

Pegylation by acylation will generally result in a poly-pegylated MGDFproduct, wherein the lysine ε-amino groups are pegylated via an acyllinking group. Preferably, the connecting linkage will be an amide. Alsopreferably, the resulting product will be substantially only (e.g.,>95%)mono, di- or tri-pegylated. However, some species with higher degrees ofpegylation (up to the maximum number of lysine ε-amino acid groups ofMGDF plus one α-amino group at the amino terminus of MGDF) will normallybe formed in amounts depending on the specific reaction conditions used.If desired, more purified pegylated species may be separated from themixture, particularly unreacted species, by standard purificationtechniques, including, among others, dialysis, salting-out,ultrafiltration, ion-exchange chromatography, gel filtrationchromatography and electrophoresis.

Alkylation

Pegylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with a protein such as MGDF in the presence of areducing agent. As with acylation, discussed above, the reactionconditions are described below.

Pegylation by alkylation can also result in poly-pegylated MGDF. Anexemplary reductive alkylation reaction with MGDF to yield apolypegylated product is shown in FIG. 15. In addition, one canmanipulate the reaction conditions as described herein to favorpegylation substantially only at the α-amino group of the N-terminus ofthe MGDF species (i.e., a mono-pegylated species). An exemplaryreductive alkylation reaction with MGDF to yield a monopegylated productis shown in FIG. 16. In either case of monopegylation or polypegylation,the PEG groups are preferably attached to the protein via a --CH₂ --NH--group. With particular reference to the --CH₂ -- group, this type oflinkage is referred to herein as an "alkyl" linkage.

Derivatization via reductive alkylation to produce a monopegylatedproduct exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available for derivatizationin MGDF. The reaction is performed at a pH (see below) which allows oneto take advantage of the pK_(a) differences between the ε-amino groupsof the lysine residues and that of the α-amino group of the N-terminalresidue of the protein. By such selective derivatization, attachment ofa water soluble polymer that contains a reactive group such as analdehyde, to a protein is controlled: the conjugation with the polymertakes place predominantly at the N-terminus of the protein and nosignificant modification of other reactive groups, such as the lysineside chain amino groups, occurs. In one important aspect, the presentinvention provides for a substantially homogeneous preparation ofmonopolymer/MGDF protein conjugate molecules (meaning MGDF protein towhich a polymer molecule has been attached substantially only (i.e.,≧95%) in a single location). More specifically, if polyethylene glycolis used, the present invention also provides for pegylated MGDF proteinlacking possibly antigenic linking groups, and having the polyethyleneglycol molecule directly coupled to the MGDF protein.

Thus, in a preferred aspect, the present invention relates to pegylatedMGDF, wherein the PEG group(s) is (are) attached via acyl or alkylgroups. As discussed above, such products may be mono-pegylated orpoly-pegylated (e.g., containing 2-6, preferably 2-5, PEG groups). ThePEG groups are generally attached to the protein at the α or ε aminogroups of amino acids, but it is also contemplated that the PEG groupscould be attached to any amino group attached to the protein, which issufficiently reactive to become attached to a PEG group under suitablereaction conditions.

The polymer molecules used in both the acylation and alkylationapproaches may be selected from among water soluble polymers or amixture thereof. The polymer selected should be water soluble so thatthe protein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. The polymer selectedshould be modified to have a single reactive group, such as an activeester for acylation or an aldehyde for alkylation, preferably, so thatthe degree of polymerization may be controlled as provided for in thepresent methods. A preferred reactive PEG aldehyde is polyethyleneglycol propionaldehyde, which is water stable, or mono C1-C10 alokoxy oraryloxy derivatives thereof (see, U.S. Pat. No. 5,252,714). The polymermay be branched or unbranched. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable. The water soluble polymer may be selected from the groupconsisting of, for example, polyethylene glycol,monomethoxy-polyethylene glycol, dextran, poly-(N-vinyl pyrrolidone)polyethylene glycol, propylene glycol homopolymers, a polypropyleneoxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g.,glycerol) and polyvinyl alcohol. For the acylation reactions, thepolymer(s) selected should have a single reactive ester group. For thepresent reductive alkylation, the polymer(s) selected should have asingle reactive aldehyde group. Generally, the water soluble polymerwill not be selected from naturally-occurring glycosyl residues sincethese are usually made more conveniently by mammalian recombinantexpression systems. The polymer may be of any molecular weight, and maybe branched or unbranched.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol, abbreviated PEG. As used herein, polyethyleneglycol is meant to encompass any of the forms of PEG that have been usedto derivatize other proteins, such as mono-(C1-C10) alkoxy- oraryloxy-polyethylene glycol.

As employed herein, MGDF is defined as including any of the variousforms of MGDF described herein. For example, full-length or truncated,glycosylated or nonglycosylated forms of MGDF are all included. Thefollowing are preferred MGDF molecules to be derivatized (in each casethe numbering refers to the amino acids numbered in accordance with FIG.11):

MGDF-1: amino acids 1-332 of SEQ ID NO: 25

MGDF-2: amino acids 1-174 of SEQ ID NO: 25

MGDF-4: amino acids 1-151 of SEQ ID NO: 25

MGDF-11: amino acids 1-163 of SEQ ID NO: 25

MGDF-12: amino acids 6-332 of SEQ ID NO: 25

MGDF-13: amino acids 6-174 of SEQ ID NO: 25

MGDF-14: amino acids 6-151 of SEQ ID NO: 25

MGDF-15: amino acids 6-163 of SEQ ID NO: 25.

The above-preferred species may be glycosylated, non-glycosylated, orde-glycosylated, preferably non-glycosylated. They may be maderecombinantly in either bacterial (e.g., E. coli) or mammalian (e.g.,CHO) cells.

The following are particularly preferred sub-groups of chemicallyderivatized molecules of this invention (in each case, they are mono- orpoly-, e.g., 2-4, PEG moieties, attached via an acyl or alkyl group):

pegylated MGDF-11 (amino acids 1-163 of SEQ ID NO: 25)

pegylated MGDF-4 (amino acids 1-151 of SEQ ID NO: 25)

pegylated MGDF-2 (amino acids 1-174 of SEQ ID NO: 25)

In general, chemical derivatization may be performed under any suitablecondition used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated MGDF willgenerally comprise the steps of (a) reacting an MGDF polypeptide withpolyethylene glycol (such as a reactive ester or aldehyde derivative ofPEG) under conditions whereby MGDF becomes attached to one or more PEGgroups, and (b) obtaining the reaction product(s). In general, theoptimal reaction conditions for the acylation reactions will bedetermined case-by-case based on known parameters and the desiredresult. For example, the larger the ratio of PEG: protein, the greaterthe percentage of poly-pegylated product.

Reductive alkylation to produce a substantially homogeneous populationof mono-polymer/MGDF protein conjugate molecule will generally comprisethe steps of: (a) reacting an MGDF protein with a reactive PEG moleculeunder reductive alkylation conditions, at a pH suitable to permitselective modification of the α-amino group at the amino terminus ofsaid MGDF protein; and (b) obtaining the reaction product(s).

For a substantially homogeneous population of mono-polymer/MGDF proteinconjugate molecules, the reductive alkylation reaction conditions arethose which permit the selective attachment of the water soluble polymermoiety to the N-terminus of MGDF. Such reaction conditions generallyprovide for pK_(a) differences between the lysine amino groups and theα-amino group at the N-terminus (the pK_(a) being the pH at which 50% ofthe amino groups are protonated and 50% are not). The pH also affectsthe ratio of polymer to protein to be used. In general, if the pH islower, a larger excess of polymer to protein will be desired (i.e., theless reactive the N-terminal αa-amino group, the more polymer needed toachieve optimal conditions). If the pH is higher, the polymer:proteinratio need not be as large (i.e., more reactive groups are available, sofewer polymer molecules are needed). For purposes of the presentinvention, the pH will generally fall within the range of 3-9,preferably 3-6.

Another important consideration is the molecular weight of the polymer.In general, the higher the molecular weight of the polymer, the fewernumber of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio. In general,for the pegylation reactions contemplated herein, the preferred averagemolecular weight is about 2 kDa to about 100 kDa (the term "about"indicating ±1 kDa). The preferred average molecular weight is about 5kDa to about 50 kDa, particularly preferably about 12 kDa to about 25kDa. The ratio of water-soluble polymer to MGDF protein will generallyrange from 1:1 to 100:1, preferably (for polypegylation) 1:1 to 20:1 and(for monopegylation) 1:1 to 5:1.

Using the conditions indicated above, reductive alkylation will providefor selective attachment of the polymer to any MGDF protein having anα-amino group at the amino terminus, and provide for a substantiallyhomogenous preparation of monopolymer/MGDF protein conjugate. The term"monopolymer/MGDF protein conjugate" is used here to mean a compositioncomprised of a single polymer molecule attached to an MGDF proteinmolecule. The monopolymer/MGDF protein conjugate will have a polymermolecule located at the N-terminus, but not on lysine amino side groups.The preparation will preferably be greater than 90% monopolymer/MGDFprotein conjugate, and more preferably greater than 95% monopolymer MGDFprotein conjugate, with the remainder of observable molecules beingunreacted (i.e., protein lacking the polymer moiety). The examples belowprovide for a preparation which is at least about 90% monopolymer/protein conjugate, and about 10% unreacted protein. Themonopolymer/protein conjugate has biological activity.

For the present reductive alkylation, the reducing agent should bestable in aqueous solution and preferably be able to reduce only theSchiff base formed in the initial process of reductive alkylation.Preferred reducing agents may be selected from the group consisting ofsodium borohydride, sodium cyanoborohydride, dimethylamine borane,timethylamine borane and pyridine borane. A particularly preferredreducing agent is sodium cyanoborohydride.

Other reaction parameters, such as solvent, reaction times,temperatures, etc., and means of purification of products, can bedetermined case-by-case based on the published information relating toderivatization of proteins with water soluble polymers (see thepublications cited herein). Exemplary details are shown in the Examplessection below.

One may choose to prepare a mixture of polymer/protein conjugatemolecules by acylation and/or alkylation methods, and the advantageprovided herein is that one may select the proportion of monopolymer/protein conjugate to include in the mixture. Thus, if desired, one mayprepare a mixture of various protein with various numbers of polymermolecules attached (i.e., di-, tri-, tetra-, etc.) and combine with themonopolymer/protein conjugate material prepared using the presentmethods, and have a mixture with a predetermined proportion ofmonopolymer/protein conjugate.

The working e

The working examples below demonstrate the preparation of chemicallymodified MGDF and the preparation of MGDF pegylated via acylation andalkylation. Thus, other aspects of the present invention relate to thesepreparations.

Generally, conditions which may be alleviated or modulated byadministration of the present polymer/MGDF include those described abovefor MGDF molecules in general. However, the polymer/MGDF moleculesdisclosed herein may have additional activities, enhanced or reducedactivities, or other characteristics, as compared to the non-derivatizedmolecules.

In yet another aspect of the present invention, provided arepharmaceutical compositions of the above. Such pharmaceuticalcompositions may contain any of the ingredients specified above fornon-derivatized MGDF molecules.

As further studies are conducted, information will emerge regardingappropriate dosage levels for treatment of various conditions in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, age and general health of the recipient, will be able toascertain proper dosing. Generally, the dosage will be between 0.01μg/kg body weight (calculating the mass of the protein alone, withoutchemical modification) and 300 μg/kg (based on the same). The preferreddose will generally be from 5 μg/kg body weight to 100 μg/kg bodyweight, particularly preferably from 10 μg/kg body weight to 75 μg/kgbody weight.

The present invention also provides a method for producing MGDF (i.e.,Mpl ligand) polypeptides or active fragments thereof. One method of thepresent invention involves introducing the cDNA encoding an Mpl ligandpolypeptide into an expression vector to make an expression system forMpl ligand. A selected host cell is transfected with the vector andcultured. The method of this present invention therefore comprisesculturing a suitable cell or cell line, which has been transfected witha DNA sequence coding on expression for an Mpl ligand polypeptide underthe control of known regulatory sequences. Regulatory sequences includepromoter fragments, terminator fragments and other suitable sequenceswhich direct/control the expression of the protein in an appropriatehost cell. The expressed factor is then recovered, isolated and purifiedfrom the culture medium (or from the cell, if expressed intracellularly)by appropriate means known to one of skill in the art. Additionally, themethods disclosed in U.S. Pat. No. 5,272,071 are also contemplated to beapplicable to the inventive polynucleotides/ polypeptides.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO) or 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. See, e.g., Gething and Sambrook, Nature 293: 620-625(1981), or alternatively, Kaufman et al., Mol. Cell. Biol., 5 (7):1750-1759 (1985) or Howley et al., U.S. Pat. No. 4,419,446. Othersuitable mammalian cell lines, are the monkey COS-1 and COS-7 celllines, and the CV-1 cell line. Further exemplary mammalian host cellsinclude primate cell lines and rodent cell lines, including transformedcell lines. Normal diploid cells, cell strains derived from in vitroculture of primary tissue, as well as primary explants, are alsosuitable. Candidate cells may be genotypically deficient in theselection gene, or may contain a dominantly acting selection gene. Othersuitable mammalian cell lines include but are not limited to, HeLa,mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHKor HaK hamster cell lines.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5α,DH10, and MC1061) are well-known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Pseudomonas spp.,other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al., Genetic Engineering 8: 277-298 (1986) and referencescited therein.

The present invention also provides recombinant molecules or vectors foruse in the method of expression of novel Mpl ligand polypeptides. Thesevectors contain the Mpl ligand DNA sequences and which alone or incombination with other sequences code for Mpl ligand polypeptides (withor without signal peptides) of the invention or active fragmentsthereof. Alternatively, vectors incorporating modified sequences asdescribed above are also embodiments of the present invention and usefulin the production of Mpl ligand polypeptides. The vector employed in themethod also contains selected regulatory sequences in operativeassociation with the DNA coding sequences of the invention and capableof directing the replication and expression thereof in selected hostcells.

One vector is pXM, which is particularly desirable for expression in COScells Y. C. Yang et al., Cell 47: 3-10 (1986)!. Another vector which isdesirable for expression in mammalian cells, e.g., CHO cells, ispEMC2B1. Mammalian cell expression vectors described herein may besynthesized by techniques well known to those skilled in this art. Thecomponents of the vectors, e.g. replicons, selection genes, enhancers,promoters, and the like, may be obtained from natural sources orsynthesized by known procedures. See, Kaufman et al., J. Mol. Biol. 159:511-521 (1982); and Kaufman, Proc. Natl. Acad. Sci. USA 82: 689-693(1985). Alternatively, the vector DNA may include all or part of thebovine papilloma virus genome Lusky et al., Cell 36: 391-401 (1984)! andbe replicated in cell lines such as C127 mouse cells as a stableepisomal element. The transfection of these vectors into appropriatehost cells can result in expression of the Mpl ligand polypeptides.

Other appropriate expression vectors of which numerous types are knownin the art for mammalian, insect, yeast, fungal and bacterial expressioncan also be used for this purpose.

The conditions to be treated by the methods and compositions of thepresent invention are generally those which involve an existingmegakaryocyte/platelet deficiency or an expected megakaryocyte/plateletdeficiency in the future (e.g., because of planned surgery). Suchconditions will usually be the result of a deficiency (temporary orpermanent) of active Mpl ligand in vivo. The generic term for plateletdeficiency is thrombocytopenia, and hence the methods and compositionsof the present invention are generally available for treatingthrombocytopenia.

Thrombocytopenia (platelet deficiencies) may be present for variousreasons, including chemotherapy and other therapy with a variety ofdrugs, radiation therapy, surgery, accidental blood loss, and otherspecific disease conditions. Exemplary specific disease conditions thatinvolve thrombocytopenia and may be treated in accordance with thisinvention are: aplastic anemia, idiopathic thrombocytopenia, metastatictumors which result in thrombocytopenia, systemic lupus erythematosus,splenomegaly, Fanconi's syndrome, vitamin B12 deficiency, folic aciddeficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome, andparoxysmal nocturnal hemoglobinuria. Also, certain treatments for AIDSresult in thrombocytopenia (e.g., AZT). Certain wound healing disordersmight also benefit from an increase in platelet numbers.

With regard to anticipated platelet deficiencies, e.g., due to futuresurgery, an Mpl ligand of the present invention could be administeredseveral days to several hours prior to the need for platelets. Withregard to acute situations, e.g., accidental and massive blood loss, anMpl ligand could be administered along with blood or purified platelets.

The Mpl ligands of this invention may also be useful in stimulatingcertain cell types other than megakaryocytes if such cells are found toexpress Mpl receptor. Conditions associated with such cells that expressthe Mpl receptor, which are responsive to stimulation by the Mpl ligand,are also within the scope of this invention.

MGDF molecules that are not themselves active in the activity assayspresented herein may be useful as modulators (e.g., inhibitors orstimulants) of the Mpl receptors in vitro or in vivo.

The polypeptides of the present invention may also be employed alone, orin combination with other cytokines, soluble Mpl receptor, hematopoieticfactors, interleukins, growth factors or antibodies, in the treatment ofthe above-identified conditions.

Therefore, as yet another aspect of the invention are therapeuticcompositions for treating the conditions referred to above. Suchcompositions comprise a therapeutically effective amount of an Mplligand polypeptide or a therapeutically effective fragment thereof inadmixture with a pharmaceutically acceptable carrier. The carriermaterial may be water for injection, preferably supplemented with othermaterials common in solutions for administration to mammals. Typically,an Mpl ligand therapeutic will be administered in the form of acomposition comprising purified protein in conjunction with one or morephysiologically acceptable carriers, excipients, or diluents. Neutralbuffered saline or saline mixed with serum albumin are exemplaryappropriate carriers. Preferably, the product is formulated as alyophilizate using appropriate excipients (e.g., sucrose). Otherstandard carriers, diluents, and excipients may be included as desired.

The present compositions can be systemically administered parenterally.Alternatively, the compositions may be administered intravenously orsubcutaneously. When systemically administered, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such pharmaceutically acceptable protein solutions, with due regardto pH, isotonicity, stability and the like, is within the skill of theart.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician, consideringvarious factors which modify the action of drugs, e.g. the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. Generally,the daily regimen should be in the range of 0.1-1000 micrograms of Mplligand protein or fragment thereof per kilogram of body weight.

The therapeutic methods, compositions and polypeptides of the presentinvention may also be employed, alone or in combination with othercytokines, soluble Mpl receptor, hematopoietic factors, interleukins,growth factors or antibodies in the treatment of disease statescharacterized by other symptoms as well as platelet deficiencies. It isanticipated that an Mpl ligand molecule will prove useful in treatingsome forms of thrombocytopenia in combination with general stimulatorsof hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocyticstimulatory factors, i.e., meg-CSF, stem cell factor (SCF), leukemiainhibitory factor (LIF), oncostatin M (OSM), or other molecules withmegakaryocyte stimulating activity may also be employed with Mpl ligand.Additional exemplary cytokines or hematopoietic factors for suchco-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5,IL-6, IL-11, colony stimulating factor-1 (CSF-1), GM-CSF, granulocytecolony stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha),IFN-beta, or IFN-gamma. It may further be useful to administer, eithersimultaneously or sequentially, an effective amount of a solublemammalian Mpl receptor, which appears to have an effect of causingmegakaryocytes to fragment into platelets once the megakaryocytes havereached mature form. Thus, administration of Mpl ligand (to enhance thenumber of mature megakaryocytes) followed by administration of thesoluble Mpl receptor (to inactivate the ligand and allow the maturemegakaryocytes to produce platelets) is expected to be a particularlyeffective means of stimulating platelet production. The dosage recitedabove would be adjusted to compensate for such additional components inthe therapeutic composition. Progress of the treated patient can bemonitored by conventional methods.

Other uses for these novel polypeptides are in the development ofantibodies generated by standard methods. Thus, antibodies that reactwith the Mpl ligands of the present invention, as well as reactivefragments of such antibodies, are also contemplated. The antibodies maybe polyclonal, monoclonal, recombinant, chimeric, single-chain and/orbispecific, etc. The antibody fragments may be any fragment that isreactive with the Mpl ligand of the present invention, such as, F_(ab),F_(ab) ', etc. Also provided by this invention are the hybridomasgenerated by presenting Mpl ligand or a fragment thereof as an antigento a selected mammal, followed by fusing cells (e.g., spleen cells) ofthe animal with certain cancer cells to create immortalized cell linesby known techniques. The methods employed to generate such cell linesand antibodies directed against all or portions of a human Mpl ligandpolypeptide of the present invention are also encompassed by thisinvention.

The antibodies may be used therapeutically, such as to inhibit bindingof the Mpl ligand and its receptor. The antibodies may further be usedfor in vivo and in vitro diagnostic purposes, such as in labeled form todetect the presence of the Mpl ligand in a body fluid.

The following examples are included to more fully illustrate the presentinvention. Additionally, these examples provide preferred embodiments ofthe invention, but are not meant to limit the scope thereof, unless soindicated. Standard methods for many of the procedures described in thefollowing examples, or suitable alternative procedures, are provided inwidely recognized manuals of molecular biology such as, for example,Sambrook et al., Molecular Cloning, Second Edition, Cold Spring HarborLaboratory Press (1987) and in Ausubel et al., (Eds), Current Protocolsin Molecular Biology, Greene associates/Wiley Interscience, New York(1990).

EXAMPLE 1 Aplastic Canine Plasma

Heparinized aplastic canine plasma ("APK9") or normal canine plasma("NK9") was produced as described in the following publications, exceptthat 450 rads of total body irradiation were delivered to recipients:

1. Mazur, E. and South, K. Exp. Hematol. 13:1164-1172 (1985).

2. Arriaga, M., South, K., Cohen, J. L., and Mazur, E. M. Blood 69:486-492 (1987).

3. Mazur, E., Basilico, D., Newton, J. L., Cohen, J. L., Charland, C.,Sohl, P. A., and Narendran, A. Blood 76: 1771-1782 (1990).

EXAMPLE 2 Human Megakaryocyte Assay

APK9 and fractionated APK9 were assayed for the ability to stimulatedevelopment of human megakaryocytes from CD34⁺ progenitor cells.CD34-selected cells were obtained from peripheral blood cells asdescribed (Hokom, M. H., Choi, E., Nichol, J. L., Hornkohl, A., Arakawa,T., and Hunt, P. Molecular Biology of Haematopoiesis 3:15-31,1994) andwere incubated in the following culture medium: Iscove's modifiedDulbecco's medium (IMDM; GIBCO, Grand Island, NY) supplemented with 1%Glutamine Pen-strep (Irvine Scientific, Santa Ana, Calif.) and 10%heparinized, platelet-poor, human AB plasma. Also included were2-mercaptoethanol (10⁻⁴ M), pyruvic acid (110 μg/ml), cholesterol (7.8μg/ml), adenosine, guanine, cytidine, uridine, thymidine,2-deoxycytosine, 2-deoxyadenosine, 2-deoxyguanosine (10 μg/ml each,Sigma); human recombinant insulin (10 μg/ml), human transferrin (300μg/ml), soybean lipids (1%, Boehringer Mannheim, Indianapolis, Ind.);human recombinant basic fibroblast growth factor (2 ng/ml, Genzyme,Cambridge, Mass.); human recombinant epidermal growth factor (15 ng/ml),platelet-derived growth factor (10 ng/ml, Amgen, Inc., Thousand Oaks,Calif.). CD34-selected cells were plated at 2×10⁵ /ml culture medium, 15ul final volume, in wells of Terasaki-style microtiter plates (Vanguard,Inc., Neptune, N.J.). Cells were incubated at 37° C. for 8 days inhumidified boxes in 5% CO₂ in air, fixed directly to the culture wellswith 1% glutaraldehyde, and incubated with a monoclonal antibodycocktail (anti-GPIb, anti-GPIIb, (Biodesign) and anti-GPIb (Dako,Carpinteria, Calif.). The immune reaction was developed with astreptavidin-beta-galactosidase detection system (HistoMark, Kirkegaardand Perry). Megakaryocytes, identified by a blue color, were countedwith an inverted phase microscope at 100× magnification. Results werepresented as the average number of megakaryocytes per well ± standarderror of the mean (SEM). In some cases, data were presented in terms of"megakaryocyte units/ml" where the degree to which a given sampleinduced megakaryocyte development was normalized to the positive APK9control for that experiment. One unit is defined as the amount ofmaterial that results in the same number of megakaryocytes as 1 ul ofAPK9 standard. Activity was accepted as due to MPL ligand if it could beblocked with 5-10 ug/ml MPL-X (soluble Mpl receptor).

APK9 has been demonstrated to contain factor(s) that stimulate humanmegakaryocyte development in this system. CD34-selected cells incubatedwith 10% NK9 for 8 days show a negligible number of blue-stainedmegakaryocytes, whereas CD34-selected cells incubated with 10% APK9 for8 days show a very large number of blue-stained megakaryocytes.

FIG. 2 shows that increasing concentrations of Mpl-X added to the humanmegakaryocyte culture system increasingly block megakaryocytedevelopment. At concentrations of Mpl-X greater than 5 μg/ml, inhibitionis complete. In this experiment, CD34-selected cells were stimulatedwith 5% APK9. This demonstrates that an activity which interacts withMpl-X (presumptive Mpl ligand) is necessary for human megakaryocytedevelopment, and implies that the Mpl ligand is present in APK9 itself.

It has been further demonstrated herein that the Mpl ligand activitynecessary for human megakaryocyte development is present in APK9. APK9(135 ml) was diluted 6-fold into Iscove's media and applied to an Mpl-Xaffinity column. Unbound material (flow through) was collected andconcentrated to the original volume before assay. Bound material waseluted in 10 ml of 1M NaCl, and 20% of the pool was diafiltered andconcentrated 4-fold for assay. CD34-selected cells incubated in mediaalone did not develop into megakaryocytes. Cells incubated in 5% APK9(same pool as applied to column) developed into 48±8 megakaryocytes perwell. Cells incubated in 10% of the unbound material did not developinto megakaryocytes. Cells incubated in 10% of the elution pooldeveloped into 120±44 megakaryocytes per well. Both the column load andthe elution pool activities were substantially completely inhibited with5 μg/ml Mpl-X in the assay.

EXAMPLE 3 Transfection of murine or human Mpl receptor into a murinecell line

A. Murine Mpl Receptor

The full length murine Mpl receptor cDNA was subcloned into anexpression vector containing a transcriptional promoter derived from theLTR of Moloney Murine Sarcoma virus. 5 μg of this construct and 1 μg ofthe selectable marker plasmid pWLNeo (Stratagene) were co-electroporatedinto an IL-3 dependent murine cell line (32D, clone 23; Greenberger etal., PNAS 80: 2931-2936 (1983)). Cells were cultured for 5 days torecover from the procedure, then incubated in selection media including800 ug/ml Geneticin (G418, Sigma) and 1 ng/ml murine IL-3. The survivingcells were then divided into pools of 2×10⁵ cells and cultured until apopulation grew out which could be further analyzed. Six populationswere tested for surface expression of Mpl receptor by FACS analysisusing a polyclonal rabbit antipeptide serum. One population was chosenfor FACS sorting using the same antipeptide serum as before. Single-cellclones of the parent cell line were selected by growth in 10% APK9 andGeneticin. After selection in APK9 for 35 days, the cells weremaintained in 1 ng/ml murine IL-3. One of the subclones, 1A6.1, was usedfor this body of work.

B. Human Mpl Receptor

The full length human Mpl receptor sequence (Vigon, I., et al., PNAS 89:5640-5644 (1992)) was subcloned into an expression vector containing thetranscriptional promoter of Moloney Murine Sarcoma virus (same vector aswith the murine receptor). Six μg of this construct and 6 μg of anamphotrophic retroviral packaging construct (Landau, N. R., Littman, D.R., J. Virology 66: 5110-5113 (1992)) were transfected into 3×10⁶ 293cells using a CaPO₄ mammalian transfection kit (Stratagene). The samecells were retransfected after 2 days and again after 4 days. The dayafter the last transfection the 293 cells were cocultivated with theIL-3 dependent murine cell line (32 D, clone 23; Greenberger et al.,PNAS 80: 2931-2936 (1983)). After 24 hours, the 32 D cells were rescuedand banded in a BSA gradient (Path-o-cyte; Miles Inc.). Cells wereexpanded in 1 ng/ml murine IL-3 and then were selected for growth in 20%APK9. Cells were sorted for cell surface expression of receptor by FACSusing a polyclonal rabbit antipeptide serum. These cells weresubsequently used in the assays.

EXAMPLE 4 1A6.1 assay for Mpl ligand

1A6.1 cells were washed free of culture IL-3 and replated (1000 cells/15μl total vol/well) in Terasaki-style microtiter plates in alpha MEM(Gibco) supplemented with 10% fetal calf serum (FCS), Geneticin (800μg/ml) and 1% pen/strep (Gibco) in 1:1 serial dilutions of test samples.After 48 hours, the number of viable cells per well was determinedmicroscopically. One unit of activity was defined as that amount ofactivity that resulted in 200 viable cells per well. Activity wasdefined as due to Mpl ligand if it could be completely blocked byincluding 5-10 μg/ml Mpl-X in the assay. Mpl ligand activity in APK9averaged 4400±539 units/ml of aplastic plasma. Unless otherwiseindicated, units of Mpl ligand activity are defined in the 1A6.1 assay.

Assays with cells transfected with the human Mpl receptor gene (Example3B) were carried out in essentially the same manner as with the 1A6.1cells.

EXAMPLE 5 Demonstration that Mpl-ligand is present in aplastic plasma orsera of mouse, dog, pig and human sources

Mpl ligand is present in the aplastic plasma or sera from murine,canine, porcine and human sources (Table 2). Plasma was collected fromBDF1 mice pre-irradiation and 12 days post-irradiation (500 rads).Plasma was tested in the 1A6.1 assay where it demonstrated 2000 units/mlactivity that was substantially completely inhibitable with Mpl-X (10ug/ml). Irradiated mouse plasma was also positive in the humanmegakaryocyte assay where it displayed an activity of 1833 units/ml.Plasma was collected from dogs pre-irradiation and 10 dayspost-irradiation (450 rads). Plasma was tested in both the 1A6.1 assayand human megakaryocyte assays. Activity was detected and completelyinhibited by Mpl-X (10 ug/ml) in both assays. Plasma was collected frompigs pre-irradiation and 10 days post-irradiation (650 rads). Plasma wastested in both the 1A6.1 assay and the human megakaryocyte assays. Inboth assays it displayed Mpl ligand activity (inhibitable by 10 ug/mlMpl-X) comparable to that found in aplastic canine plasma. Sera fromaplastic humans was obtained. This material was collected from bonemarrow transplantation patients. The sera from 6 patients were assayedin the 1A6.1 assay where it showed an activity of 903 units/ml, 88% ofwhich was due to Mpl ligand (inhibitable with 10 ug/ml Mpl-X). Sera from14 aplastic patients has also been tested in the human megakaryocyteassay. As a group, they displayed substantial activity, 941 megunits/ml, which was completely inhibitable with 10 ug/ml Mpl-X. MurineIL-3 data is included to demonstrate the specificity of the 1A6.1 assay.Although this recombinant cytokine induces growth of the cell line, itis not blocked by 10 ug/ml Mpl-X.

                  TABLE 2                                                         ______________________________________                                                1A6.1 Cell Assay                                                                            Human Meg Assay                                                 (units/ml)    (meg units/ml)                                          Species   media     +Mpl-X    media   +Mpl-X                                  ______________________________________                                        Normal mouse                                                                            0+/-0     0+/-0     0+/-0   0+/-0                                   Aplastic mouse                                                                          2000      0         1833    not done                                Normal canine                                                                           0+/-0     0+/-0     0+/-0   0+/-0                                   Aplastic canine                                                                         4400+/-539                                                                              0+/-0     792+/-128                                                                             0+/-0                                   Normal porcine                                                                          0+/-0     0+/-0     0+/-0   0+/-1                                   Aplastic porcine                                                                        3866+/-1136                                                                             0+/-0     1284+/-182                                                                            10+/-10                                 Normal human                                                                            0+/-0     0+/-0     0+/-0   0+/-0                                   Aplastic human                                                                          903+/-64  114+/-33  941+/-178                                                                             0+/-0                                   murIL3    6000+/-565                                                                              6000+/-565                                                                              not done                                                                              not done                                ______________________________________                                    

EXAMPLE 6 Mpl ligand stimulates 1A6.1 cell growth and humanmegakaryocyte development

Mpl ligand (enriched at least about 100,000-fold after lectin andaffinity chromatography procedures; see Example 7) stimulates the growthof the 1A6.1 cell line and the development of human megakaryocytes fromCD34-selected peripheral blood cells in a dose-dependent manner. Theactivity responsible is due to Mpl ligand as shown in FIGS. 2 and 3since the activities in both assays can be completely blocked withMpl-X.

It has also been shown by the inventors that FACS purified peripheralblood CD34⁺ cells, when incubated in Mpl ligand (100 units/ml for 9 daysin this case), develop into phenotypically normal, maturemegakaryocytes. This establishes that purified Mpl ligand has the sameeffect on megakaryocytes as does crude APK9. Furthermore, thisexperiment used purified CD34⁺ cells (100% CD34⁺) as opposed toCD34-selected cells which are generally only 30-50% CD34⁺.

EXAMPLE 7 Purification of Canine Mpl Ligand

I. Summary

Proteins (25 kd and 31 kd) that display activities predicted for aligand for the Mpl receptor were purified. The proteins were purifiedfrom the plasma of irradiated dogs by a scheme employing wheat germagglutinin (WGA) affinity chromatography, Mpl receptor affinitychromatography, anion exchange chromatography, gel filtrationchromatography, and C4 reversed phase HPLC. See, FIG. 4 for an overviewof this purification scheme. The 25 kd and 31 kd Mpl ligands have beenhighly purified to apparent homogeneity and have been determined tocontain the amino acid sequences disclosed herein.

II. Methods

A. Clarification of plasma.

Frozen plasma (a total of 20 liters) from irradiated dogs (seeExample 1) was thawed overnight at 4° C.; thawing of larger bottles wasinitiated at room temperature for several hours before placement in thecold room. Insoluble material was removed by centrifugation for 6 hoursat 11,000×g. The plasma was diluted with phosphate buffered saline, pH7.3, containing 0.01% sodium azide (PBS/azide) and filtered through a1.2 μm filter. The clarification procedure typically resulted in anapproximate two-fold dilution of the starting material.

B. Wheat Germ Agglutinin Affinity Chromatography.

All operations were carried out at 4° C. The clarified plasma (in twobatches) was applied to a column of immobilized wheat germ agglutinin (1liter, 10×12 cm, E Y Laboratories), equilibrated in PBS/azide. Aftersample application, unbound material was washed from the column withPBS/azide, followed by a wash with 0.5M NaCl in 20 mM Tris-HCl, pH 8.Mpl ligand activity, bound by the WGA column, was eluted with 0.35MN-acetylglucosamine (GlcNAc), 0.5M NaCl, 20 mM Tris-HCl, pH 8. Mplligand activity could not be detected in the flow through or washfractions.

C. Mpl-X receptor affinity chromatography.

The soluble murine Mpl receptor (Mpl-X) that was used corresponded tothe entire extracellular domain of the Mpl receptor minus Trp atposition 483 (See Vigon, et al, i 8: 2607-2615 (1993)). In order tooptimize binding of Mpl ligand to the Mpl-X receptor affinity column,the WGA elution pool was concentrated using a membrane ultrafilter(10,000 molecular weight cut off, YM-10, Amicon) and NaCl adjusted to0.2M by subsequent dilution. The concentrated WGA pool was applied to a20 ml m-Mpl-X (murine Mpl soluble receptor)/CNBr activated Sepharosecolumn (2.6×4.2 cm, 1.5 mg m-Mpl-X per ml of resin) at a flow rate 0.9ml/min. The column was washed with 40 ml of PBS/azide at 1.5 ml/min,followed by a high salt wash (405 ml) with 10 mM Tris-HCl, 1M NaCl, 1 mMCHAPS, pH 8.0. The column was then eluted with 20 mM CAPS, 1M NaCl, 5 mMCHAPS, pH 10.5. Appropriate fractions were collected. Tris was added toeach fraction to neutralize the pH.

Both the SDS-PAGE and the absorbance at 280 nm of the elution profile ofan Mpl-X receptor affinity column reveal an early protein peak infractions 1-4, while the majority of the Mpl ligand activity elutedafter fraction 5.

D. Mono-O Anion exchange chromatography.

The highest purity fractions from several Mpl-X receptor affinitycolumns were combined, concentrated, and diafiltered against 20 mMTris-HCl, 5 mM CHAPS, pH 8.7 to a final volume of 58.5 ml. The proteinconcentration of the pool was estimated by absorbance at 280 nm to be0.12 mg/ml (approximately 7 mg total protein). The pool was loaded at0.5 ml/min onto a Mono Q HR 5/5 column (Pharmacia) equilibrated in 20 mMTris-HCl, 5 mM CHAPS, pH 8.7. The column was eluted with a lineargradient to 0.36M NaCl in the same buffer over 27 minutes. The columnwas then washed with a 6 minute gradient to 0.54M NaCl, and finally witha step wash at 0.9M NaCl. One ml fractions were collected.

The elution profile of the Mono Q column shows that no Mpl ligand, andnegligible protein, could be detected in the flow-through and washfractions. Much of the Mpl ligand activity elutes in fractions 5-7,during the initial stages of the NaCl gradient. A "shoulder" of activityis observed in fractions 8-10, followed by a second major peakcomprising fractions 11-15.

A distinct 25 kd band is observed by SDS-PAGE (nonreducing) in theactive fractions. The intensity of the band directly corresponds withthe Mpl ligand activity in the fractions. The band was absent infractions 3 and 4 (no activity). It was prominent in fractions 5 and 6(1A6.1 activity peak) and a similar, intensely stained band, was presentin fractions 11-14 (1A6.1 activity peak). The band is faint in the poolof fractions 15 and 16, corresponding with the significantly loweractivity in fraction 16.

E. Gel Elution Experiments.

Gel elution experiments were performed using aliquots of Mono Qfractions 5 and 6 or Mono Q fractions 13 and 14. For these experiments,pools of fractions 5 and 6 (6 μl each) or 13 and 14 (7.5 μl each) weremade, mixed with SDS-PAGE sample buffer (nonreducing), and applied to12% SDS gels. Upon completion of electrophoresis, lanes of interest weresliced (1 mm) and the slices were diced into small pieces with razorblades. The pieces were transferred to 1.5 ml microfuge tubes containing0.5 ml PBS/5mM CHAPS and gently agitated overnight at 4° C. The next daythe tubes were spun briefly, an aliquot was removed, and the sample wasdiafiltered against Iscove's medium supplemented with BSA as a carrierprotein. The diafiltered samples were submitted for assay.

The results reveal that two peaks of Mpl ligand activity can beobserved. One peak corresponds to the 25 kd region of the gel, while asecond peak of Mpl ligand activity is observed in the 31 kd region.

F. Superdex 200 Gel Filtration.

Fractions 13-16 from the Mono Q anion exchange column, as well as twoequivalent fractions from a second Mono Q fractionation, were combinedand concentrated using a membrane ultrafilter (Centricon-10, Amicon).SDS was added to a final concentration of 0.1%, and the sample wasinjected onto a Superdex 200 HR 10/30 (Pharmacia) column. The column wasequilibrated in 50 mM Tris-HCl, 0.1% SDS, pH 7.5 at a flow rate of 0.3ml/min, and was operated at room temperature. One minute fractions werecollected. The results were that most of the protein in the sampleelutes in fractions 32-40, while the Mpl ligand activity is detected infractions 42-46. Analysis of fractions SDS-PAGE showed a distinct 25 kdband in the active fractions.

G. C4 Reversed Phase HPLC.

Superdex 200 fractions 43-46 combined or fraction 42 alone wereconcentrated using a membrane ultrafilter (Microcon-10, Amicon). Theconcentrated pools were separately applied to a 1×100 mm C4 reversedphase microbore column (SynChropak RP-4). The column was equilibrated in0.04% TFA in water (A Buffer); B Buffer was 0.035% TFA in 80%acetonitrile. After injection of the sample, a linear gradient to 45% Bover 4 min was performed, followed by a linear gradient to 75% B over 40min. Flow rate was 75 μl/min. The results of purification of fraction 42are presented in FIG. 5. Distinct Mpl ligand activity peaks wereobserved in fractions 21-23. These fractions were analyzed on a 14%polyacrylamide gel under nonreducing and reducing conditions. Fraction21 was composed of a single 31 kd band; fraction 23 was composed of asingle, broad 25 kd band; and fraction 22 contained bands in both the 25kd and 31 kd regions. No other significant bands were visible. Note thatearlier gel elution experiments had ascribed Mpl ligand activity to bothof these regions. A single, minor high molecular weight band wasobserved in all fractions of the nonreducing gel, but could not be seenin the reducing gel.

H. N-terminal Sequence Analysis of 25 kd and 31 kd Mpl ligands.

N-terminal sequence analysis was carried out on C4 RP-HPLC fractionscontaining activity. The sequences determined for these proteins arereported above. In addition to the major sequence corresponding to the25 kd band (at least 90% of the total of the applied sample), sequencingdetected two minor sequences (which were associated with the minorcontaminating band described in part G above). Comparisons with knownsequences revealed that the minor sequences were canine Ig heavy chainand kappa chain. If desired, these minor impurities could be furtherreduced in quantity by application of another purification step, such aspreferably another gel filtration step.

I. Comparison of Mpl ligand activities in the C4 purified fractions

FIG. 6 shows data demonstrating that the activities present in fractions22 and 23 from the C4 RP-HPLC chromatography step are substantiallyequivalent. Fraction 22 contained a mixture of the 25 and 31 kd bands,whereas fraction 23 contained only the 25 kd band. Aliquots of eachfraction were diluted 1:45000. The diluted fractions stimulated 1A6.1cell growth substantially equally, (fraction 22, 5400 cells per well;fraction 23, 6000 cells per well). The diluted fractions were incubatedwith increasing concentrations of Mpl-X. The fractions were equallysensitive to inhibition by Mpl-X, both being completely blocked with7-1.4 ug/ml. This indicates that the active protein(s) in each fractionare Mpl ligand species with equivalent biological activity.

EXAMPLE 8 Comparison of Mpl ligand to other factors on megakaryocytedevelopment

A number of recombinant factors or organic compounds such as phorbolmyristic acetate (PMA) have been reported to impact megakaryocyte growthor development. Accordingly, the effects of these factors onCD34-selected peripheral blood cells were investigated. Humanrecombinant interleukin 3 (IL-3, 1-2 ng/ml), stem cell factor (SCF, 50ng/ml), interleukin 6 (IL-6, 25 ng/ml), erythropoietin (EPO, 1 Unit/ml),leukemia inhibitory factor (LIF, 10 ng/ml), and granulocyte-macrophagecolony-stimulating factor (GM-CSF, 25 ng/ml, Amgen, Inc.); interleukin11 (IL-11, 25 ng/ml, R+D Systems, Minneapolis, Minn.); phorbol myristicacetate (PMA, 10⁻¹⁰ M, Sigma) were added to cultures as indicated. Mplligand was used at 275 units per ml, APK9 was used at 5% (equivalent to220 units/ml). Factors tested in combination were at the sameconcentration as when tested individually. After 8 days in culture, thecells were fixed directly in the wells and stained for megakaryocytes(n=6 wells per condition) or counted for total cell number (n=3 wellsper condition). Data are presented as mean ± SEM.

FIG. 7 shows that APK9 and Mpl ligand resulted in the greatest number ofmegakaryocytes per well. IL-3 also resulted in megakaryocytedevelopment, especially in combination with SCF. IL-6, IL-11, or EPO hadlittle effect on megakaryocyte numbers either alone or in combinationwith IL-3. PMA, LIF and GM-CSF had little effect. In FIG. 8 are datafrom the same experiment showing the total number of cells found perwell ("cellularity"). APK9 and Mpl ligand had little effect oncellularity while IL-3 and SCF had modest effects. SCF and IL-3 incombination had the greatest effects. The data shown in FIGS. 7 and 8were used to calculate percentages of megakaryocytes per well, as shownin FIG. 9. Clearly, the factor which results in the greatest percentageof megakaryocytes per culture well is Mpl ligand, the active ingredientin APK9. This is indicative of the specificity of Mpl ligand towardsmegakaryocytes.

EXAMPLE 9 The megakaryocyte promoting activity of Mpl ligand is notdependent on human IL-3

Mpl ligand stimulates the development of human megakaryocytes when usedas a supplement to the culture medium described in Example 2. AlthoughIL-3 is not an ingredient of the medium, it could be present inundetectably low levels in the normal human plasma present in themedium. However, even if present, IL-3 is not involved in Mplligand-induced megakaryopoiesis. This is shown in FIG. 10. IL-3 at 2ng/ml contains an activity in the human meg assay of 14,900 megunits/ml. This activity is 97% inhibited with anti-IL-3 (3.3 ug/ml;Genzyme, Cambridge, Mass.). MPL ligand at 8203 meg units/ml was notinhibited with anti-IL-3.

EXAMPLE 10 Analysis of porcine Mpl ligand

I. Summary

Proteins from irradiated pig plasma with Mpl ligand activity werecharacterized with WGA affinity chromatography, Mpl receptor affinitychromatography, ion exchange chromatography and C4 reverse phase HPLC.The activity was also characterized by elution from slices fromSDS-polyacrylamide gels.

    ______________________________________                                        Chromatography Comments                                                       ______________________________________                                        WGA affinity column                                                                          4.4 × 10.sup.6 units applied.                                           3.4 × 10.sup.6 units recovered                           Mpl receptor column                                                                          2.7 × 10.sup.6 units applied                                            2.4 × 10.sup.6 units recovered                           Mono S ion exchange                                                                          2.4 × 10.sup.6 units applied                             pH 6.0         4.4 × 10.sup.6 units recovered                           C4 reverse phase                                                                             Activity recovered fractions 23-25                             HPLC                                                                          Gel elution    Two activities clearly                                         Experiments    distinguished, one at approximately                                           18 kd, the other at approximately 28                                          kd.                                                            ______________________________________                                    

EXAMPLE 11 Cloning of the Human Mpl-ligand, Human MGDF

Two approaches are outlined in the following:

I. First Exemplary Cloning Approach

A. Generation of human MDGF probe

A number of degenerate PCR primers were designed based on the aminoterminus sequence of the canine protein. Different primer pairs wereused to amplify the MGDF gene from the human genomic DNA. After 40cycles of amplification, using the sense primers 5' GCN CCN CCN GCN TGYGA 3'(SEQ ID NO: 4), encoding the first five amino acids of the canineprotein (SEQ ID NO: 1) and the antisense primer: 5' GCA RTG YAA CAC RTGNGA RTC 3'(SEQ ID NO: 5), encoding amino acids 16 to 21 of SEQ ID NO: 1,the PCR product was run on a 2.0% agarose gel in TBE buffer.

The 63 bp band was cut out from the agarose gel and reamplified usingthe same primer set. The PCR product was cloned in the PCR II vector(Invitrogen, San Diego). A number of colonies were screened by DNAsequencing. The plasmid DNA encoding a peptide similar to the canineMGDF protein was used as the source to generate a radioactive probe toscreen the cDNA libraries. The amino acid sequence encoded by the genefragment is as follows:

Ala-Pro-Pro-Ala-Cys-Asp-Leu-Arg-Val-Leu-Ser-Lys-Leu-Leu-Arg-Asp-Ser-His-Val-Leu-His(SEQ ID NO: 6)

The agarose band containing the human MGDF was used to generate theprobe by hot PCR. A typical PCR reaction of 100 μl contained thefollowing ingredients:

    ______________________________________                                        template DNA           2-3    μl                                           5' primer (SEQ ID NO:4)                                                                              1      μl, 20 pmoles                                3' primer (SEQ ID NO:5)                                                                              1      μl, 20 pmoles                                10 X buffer            10     μl                                           dATP (0.1 mM)          2      μl                                           dTTP (10 mM)           2      μl                                           dGTP (10 mM)           2      μl                                           dCTP (0.1 mM)          2      μl                                           dCTP, p.sup.32 (10 uC/ul)                                                                            5      μl                                           dATP, p.sup.32 (10 uC/ul)                                                                            5      μl                                           Taq DNA polymerase     0.5    μl, 2.5 units                                water                  77     μl                                           total volume           100    μl                                           ______________________________________                                    

The amplification conditions were as follows

initial heating 94° C., 2 min

anneal 53° C., 30 sec

extension 72° C., 30 sec

denaturation 94° C., 30 sec.

40 cycles of amplification was carried out in a Perkin Elmer GeneAmpSystem 9600.

The product was purified by passing through a push column (Stratagene,San Diego). One 1 μl of the probe was counted in a scintillationcounter. Probes containing 1 to 3 million counts per ml were added tothe hybridization mix.

B. Construction of fetal liver library

Human fetal liver polyA⁺ RNA was purchased from Clontech laboratories.About 4 μg of RNA was used for cDNA synthesis, in which priming wascarried out using a random hexamer, 5' GTA CGC GTT CTA GAN NNN NNT 3',(SEQ ID NO: 7) attached to an oligo containing an Xba I site.

The Gibco-BRL protocol was used to generate the double stranded cDNA.The Eco R I-Bst X I adaptor (Invitrogen, San Diego) was ligated to thedouble stranded cDNA, followed by digestion with the restriction enzyme,Xba I. Size selection of the cDNA was carried out on a S500 Sephacrylcolumn (Life Technologies, Inc.). cDNAs larger than 400 bp were ligatedto the mammalian expression vector v19.8 (Martin, F., Cell 63: 203-211(1990)) which was already digested with Eco RI and Xba I. Competent E.coli DH10 cells were transformed and the resulting cDNA library wassplit into 7 pools of 100,000 cDNA each.

C. Screening the lambda library

A human fetal kidney library in lambda gt11 was bought from Clontechwith a titer of 650 million pfu/ml. About 2 million plaques werescreened with a probe generated by PCR (see above). Hybridization wasdone in 6×SSC, 5×Denhardt, 0.1% SDS, 100 μg/ml single strand salmonsperm DNA for 15 hours at 56° C.

Multiple rounds of screening were carried out. DNA was amplified fromsingle plaques and hybridized with the internal primer 5' AGT TTA CTGAGG ACT CGG AGG 3'(SEQ ID NO: 8) encoding amino acids 7 to 13 in SEQ IDNO: 6 to identify the true positives.

D. 3 prime Rapid Amplification of cDNA Ends (RACE)

Polyadenylated RNA from human fetal kidney and fetal liver were boughtfrom Clontech. One microgram RNA was reverse transcribed using the oligo5' TTC GGC CGG ATA GGC CTT TTT TTT TTT TTT 3'(SEQ ID NO: 9) as theprimer.

The Gibco-BRL cDNA synthesis kit (Life Technologies Inc., Cat.#18267-013) was used to generate the first strand cDNA. The final volumewas 30 μl. The reaction was stopped by adding 500 mM EDTA to a finalconcentration of 10 mM and kept at -20° C.

For initial PCR, 0.5 μl of cDNA was used as the template per reaction.The primer SEQ ID NO: 9 and the competitor oligo 5' TTC GGC CGG ATA GGCCTT TTT TTT TTT TT-P 3'(SEQ ID NO: 10) were used as the antisenseprimers, while the oligonucleotide 5' TGC GAC CTC CGA GTC CTC AG 3'(SEQID NO: 11) encoding amino acids 5 to 11 of SEQ ID NO: 6, was used as thesense primer. Forty cycles of amplification were carried out using thefollowing protocol: 94° C., 30 sec; 65° C., 30 sec; 72° C., 30 sec,after an initial 2 min incubation at 94° C. A Perkin Elmer GeneAmpSystem 9600 was used for amplification.

Nesting was carried out using the sense primer 5' GAG TCC TCA GTA AACTGC TTC GT 3'(SEQ ID NO: 12) encoding amino acids 8 to 14 of SEQ ID NO:6, while SEQ ID NO: 9 and SEQ ID NO: 10 served as the antisense primers.Forty cycles of amplification were carried out with annealing at 65° C.The PCR products were run on a 0.8% agarose gel and then photographedunder UV light. Bands around 0.8 to 1.2 kb were visible.

The PCR products were then cloned in the vector PCR II (Invitrogen).Individual colonies were picked and plasmids were isolated using theQiagen kits cat #12143 and 12145. Double stranded dye primed sequencingwas done using the vector primers. The sequences were analyzed byvarious types of GCG software.

E. 5' and 3' primer extension

In order to isolate the sequence of the full length MGDF gene, 3' and 5'primer extensions were carried out using different pools of fetal liverlibrary as the template. For the amplification of the 5 primer of thecDNA, about 20 ng of cDNA from each pool was used as the template. AMGDF specific antisense primer 5' GGA GTC ACG AAG CAG TTT AC 3'(SEQ IDNO: 13) encoding amino acids 12 to 17 of SEQ ID NO: 6 and the 5' vectorv19.8 sense primer 5' CCT TTA CTT CTA GGC CTG 3'(SEQ ID NO: 14) wereused. Amplification was carried out for 30 cycles with annealing at 53°C. Nesting was done for 30 cycles with the antisense primers 5' GAG GTCACA AGC AGG AGG A 3'(SEQ ID NO: 15) encoding amino acids 1 to 6 of SEQID NO: 6 and the vector primer SEQ ID NO: 14.

For the primer extension of the 3' ends of the MGDF cDNAs, the antisensevector primer 5' GGC ATA GTC CGG GAC GTC G 3'(SEQ ID NO: 16) and theMGDF specific primer 5' TCC TCC TGC TTG TGA CCT C 3'(SEQ ID NO: 17)encoding amino acids 1 to 6 of SEQ ID NO: 6, were used. Amplificationwas carried out for 30 cycles with annealing at 58° C.

Nesting amplification for 30 cycles was done using the MGDF primer SEQID NO: 12 and the vector primer SEQ ID NO: 16. Specific bands appearedin pool numbers 1, 7 and 8, which were cloned in the PCR II vector.Purified plasmid DNA from single colonies was purified and sequenced.

F. Isolation of full length clones of human MGDF

Many of the initial clones lacked part of the amino terminus of MGDF,since part of the MGDF sequence was used for priming and nesting. Primer5' CCA GGA AGG ATT CAG GGG A 3'(SEQ ID NO: 18), whose sequence wasobtained from the 5 primer extension experiments as described above wasused as the sense primer. The vector primer SEQ ID NO: 16 served as theantisense primer. 35 cycles of amplification was carried out withannealing at 58° C. MGDF specific primer 5' CAA CAA GTC GAC CGC CAG CCAGAC ACC CCG 3'(SEQ ID NO: 19) with a Sal I site and the vector primer(SEQ ID NO: 15) were used for nesting for 35 cycles. The PCR product wascloned in PCR II vector and sequenced.

II. Second Exemplary Cloning Approach

A. Cloning Of Canine MGDF N-Terminus cDNA

Degenerate oligonucleotide primers were designed based on the canineMGDF N-terminus amino acid sequence described in the previous sectionand used as primers in polymerase chain reactions (PCRs) to amplifyMGDF-encoding cDNA sequences. Total RNA was prepared from canine kidneysamples by the guanidinium isothiocyanate method of Chomzynski andSacchi (Biochem. 162: 156-159 (1987)). First strand cDNA was preparedwith a random primer-adapter 5' GGC CGG ATA GGC CAC TCN NNN NNT 3' (SEQID NO: 20) using MoMULV reverse transcriptase and used as template insubsequent PCRs.

PCR was performed on 0.5 microliters, about 50 ng, of the cDNA, usingPrimer A 5' GCN CCN CCN GCN TGY GA 3' (SEQ ID NO: 4), a sense strandprimer encoding amino acids 1-6 of SEQ ID NO: 1, and either primer B 5'GCA RTG NAG NAC RTG NGA RTC 3' (SEQ ID NO: 5) or primer C 5' GCA RTG YAANAC RTG NGA RTC 3' (SEQ ID NO: 21), which are antisense strand primersencoding amino acids 16-21 of SEQ ID NO: 1 with three extra nucleotidesat the 5' termini to increase annealing stability. PCR with Taqpolymerase was performed for 35 to 45 cycles, until product bands wereapparent on agarose gel electrophoretic analysis. For the first twocycles of PCR, the reannealing step was performed at 37° C. for 2minutes; for the remainder of the cycles reannealing was at 50° C. for 1minute. Multiple product bands were observed in each reaction. Portionsof the gel containing bands of approximately the expected size (66 bp)were collected with the tip of a Pasteur pipette and re-amplified withthe same primer pair. The DNA products were cloned into vector PCR II(Invitrogen) according to the manufacturer's instructions. Three cloneswere sequenced and were found to encode, in one reading frame, theexpected canine MGDF sequence, residues 1-21. In this way unique canineMGDF cDNA sequence was obtained spanning the region from the thirdnucleotide of codon 6 through the third nucleotide of codon 15. One ofthese clones served as the template for preparation of a labeled canineMGDF cDNA probe.

B. Construction of cDNA library from human fetal liver

RNA has been isolated from human fetal Liver (International Institutefor the Advancement of Medicine, Exton, Pa.) by lysis of tissue in 5.5Mguanidinium thiocyanate and purification via CsTFA (Pharmacia)centrifugation. Polyadenylated RNA was selected using oligo (dT)₂₅dynabeads (Dynal, according to manufactures instruction). Doublestranded cDNA was produced from this RNA using Superscript plasmidsystem for cDNA synthesis (Life Technologies, Inc.) except a differentlinker adapter: 5' TTG GTG TGC ACT TGT G 3' (SEQ ID NO: 22) and 5' CACAAG TGC ACA CCA ACC CC 3'(SEQ ID NO: 23), was used. After size selectionthis cDNA was directionally inserted into the Bst XI and Not I sites ofthe mammalian expression vector pBCB (pBCB is derived from the plasmidRc/CMV, Invitrogen, comprising the puc19 backbone, CMV promoter and BGHpolyadenylation site). The ligated DNA was electroporated into electrocompetent bacterial strain 10B (Life Technologies, Inc.).

C. Screening of human fetal liver cDNA library for MGDF

Filter replicas of the human fetal liver library were hybridized toradioactively labeled canine MGDF N-terminus cDNA PCR product (5×SSPE,2×Denhardt's, 0.05% Na pyrophosphate, 0.5% SDS, 100 μg/ml yeast tRNAlysate and 100 μg/ml denatured salmon sperm DNA) at 64° C. for 18 h.Filters were washed at 64° C. in 5×SSPE, 0.5% SDS and exposed overnight. Two different clones hybridizing to this probe were isolated andanalyzed.

D. Expression of human MGDF cDNA clones

Purified DNA from MGDF cDNA clones was transfected into 293 EBNA cells(Invitrogen). 1.5 μg of DNA was mixed with 7.5 ul Lipofectamine (LifeTechnologies, Inc.) in 100 ul of serum free DMEM. After a 20 minuteincubation at room temperature the DNA-Lipofectamine mixture was addedto 5×10⁵ cells/well (24 well square Greiner plate) in 400 ul DMEM, 1%serum (Fetal Clone II) and incubated for 6 hours at 37° C. 500 ul DMEM,20% serum (Fetal Clone II) was added to the cells. 16 hours later themedia was aspirated and 500 ul DMEM, 1% serum (Fetal Clone II) wasadded. 72 hours later the conditioned media were collected andcentrifuged through a 0.22 micron spin-filter. The conditioned mediawere assayed for MGDF biological activity.

III. Description and Activity of Human MGDF Clones

Based on the above-described cloning strategies, the human cDNA clonesshown in FIG. 11 and FIG. 12 were obtained. Each of these sequences inthe Figures contains a putative signal sequence of amino acids -21 to-1, so the mature proteins start at amino acid 1 in each case.

The results of activity assays using the cell-based assay described inExample 4A above with MGDFs 1-3 are presented in Tables 3 and 4 below.In Table 3, conditioned media from 293 EBNA cells transfected with eachconstruct was collected after 2 days of culture then tested on 1A6.1cells (32D/mur-MPL+) ±10 ug/ml mur-MPL-X. In Table 4, conditioned mediafrom 293 EBNA cells transfected with each construct was collected after4 days of culture then tested on both 32D/mur-MPL+ cells (Example 3A)and 32D/hu-MPL+ cells (Example 3B). As can be seen, human MGDF-1 (aminoacids 1-332 of SEQ ID NO: 25) and MGDF-2 (amino acids 1-174 of SEQ IDNO: 25) but not MGDF-3 (amino acids 1-265 of SEQ ID NO: 27) were foundto be active on cell lines expressing both the murine and human forms ofMpl. The cell line expressing the human MPL receptor is more responsiveto human MGDF-1 (amino acids 1-332 of SEQ ID NO: 25) and MGDF-2 (aminoacids 1-174 of SEQ ID NO; 25) than is the cell line expressing themurine Mpl receptor.

                  TABLE 3                                                         ______________________________________                                                           U/ml       U/ml                                            Clone              (-mur-MPL-X)                                                                             (+mur-MPLX)                                     ______________________________________                                        Media                 0       0                                               PBCO (control plasmid)                                                                              0       0                                               MGDF-1             12,800     800                                             (amino acids 1-332 of SEQ ID NO:25)                                           MGDF-1 12,800      566                                                        (amino acids 1-332 of SEQ ID NO:25)                                           (repeat)                                                                      MGDF-2               4525     400                                             (amino acids 1-174 of SEQ ID NO:25)                                           MGDF-2              12800     1131                                            (amino acids 1-174 of SEQ ID NO:25)                                           (repeat)                                                                      MGDF-3                0       0                                               (amino acids 1-265 of SEQ ID NO:27)                                           MGDF-3                0       0                                               (amino acids 1-265 of SEQ ID NO:27)                                           (repeat)                                                                      APK9 control       4400 +/- 400                                                                             0                                               ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                                         U/ml        U/ml                                             Clone            32D/mur-MPL+                                                                              32D/hu-MPL+                                      ______________________________________                                        MGDF-1           1600        25,600                                           (amino acids 1-332 of                                                         SEQ ID NO:25)                                                                 MGDF-2           6400        50,000                                           (amino acids 1-174 of                                                         (SEQ ID NO:25)                                                                MGDF-2 (amino acids                                                                            6400        50,000-100,000                                   1-174 of (SEQ ID NO:25)                                                       (repeat)                                                                      ______________________________________                                    

The following Table 5 shows that the activities of human MGDF-1 (aminoacids 1-332 of SEQ ID NO: 25) and MGDF-2 (amino acids 1-174 of SEQ IDNO: 25) on 32D/hu-MPL+ cells (Example 3B) are substantially completelyinhibited by soluble human mpl receptor (hu-MPL-X). Hu-MPL-X was presentas conditioned media collected from CHO cells producing the protein. TheCHO hu-MPL-X conditioned media was concentrated 120-times then added tothe cultures at 6.6%. Conditioned media from control CHO cultures had noeffect on the assay. The assay was carried out as described in Example4B except that the viable cells were assessed after 3 days.

                  TABLE 5                                                         ______________________________________                                                           U/ml       U/ml                                            Clone              (- Hu-MPL-X)                                                                             (+Hu-MPL-X)                                     ______________________________________                                        MGDF-1             530        0                                               (amino acids 1-332 of SEQ ID NO:25)                                           MGDF-2             270        0                                               (amino acids 1-332 of SEQ ID NO:25)                                           ______________________________________                                    

Human Megakaryocyte Assay

MGDF-1 (amino acids 1-332 of SEQ ID NO: 25) and MGDF-2 (amino acids1-174 of SEQ ID NO: 25) but not MGDF-3 (amino acids 1-265 of SEQ ID NO:27) induced the formation of megakaryocytes from peripheral bloodCD34-selected cells. The experiment described in Table 6 was performedessentially as described in Example 2 except that peripheral blood cellswere CD34-selected without elutriation and the culture was harvestedafter 7 days. Conditioned media from each 293 EBNA MGDF construct wasused at 20% final volume ±30 μg/ml mur-MPL-X. APK9 control was used at6% final volume.

                  TABLE 6                                                         ______________________________________                                                    Megakaryocytes per                                                                          Megakaryocytes per                                  Clone       Well (-mur-MPL-X)                                                                           Well (+mur-MPL-X)                                   ______________________________________                                        vector      0             0                                                   control                                                                       APK9        100 +/- 3     0                                                   control                                                                       MGDF-1       142 +/- 48   17 +/- 2                                            (amino acids 1-332                                                            of SEQ ID NO:25)                                                              MGDF-2      100 +/- 3      6 +/- 2                                            (amino acids 1-174                                                            of SEQ ID NO:25)                                                              MGDF-2       86 +/- 10    0                                                   (amino acids 1-174                                                            of SEQ ID NO:25)                                                              repeat                                                                        MGDF-3A      2 +/- 2      0                                                   (amino acids 1-265                                                            of SEQ ID NO:27)                                                              ______________________________________                                    

EXAMPLE 12

The following example describes the synthesis of 12 different pegylatedMGDF molecules, PEG 9-PEG 12 and PEG 14-PEG 21. In each case, the MGDFmolecule that was pegylated was E. coli derived MGDF-11 (amino acids1-163 of SEQ ID NO: 25) (amino acids 1-163, numbering from the beginningof the mature protein). Details regarding all of these pegylated speciesare summarized in Tables 7-10 below.

12.1 Preparation of poly-MePEG-MGDF conjugates by MGDF acylation withactivated MePEG derivatives

Preparation of poly-MePEG(20 kDa)-MGDF conjugate (PEG 11).

A cooled (4° C.) solution of MGDF (2.5 mg/ml) in 0.1M BICINE buffer, pH8, was added to a 10-fold molar excess of solid MePEG succinimidylpropionate (MW 20 kDa) (Shearwater Polymers, Inc.). The polymer wasdissolved by gentle stirring and the reaction further conducted at roomtemperature.

The extent of the protein modification during the course of the reactionwas monitored by size exclusion (SEC) HPLC using Superdex 200 HR 10/30column (Pharmacia Biotech) eluted with 0.1M sodium phosphate buffer pH6.9 at 0.7 ml/min.

SEC HPLC analysis of the reaction mixture at the 30 minute time pointindicated that no free protein was left in the reaction mixture. At thispoint the protein concentration in the reaction mixture was reduced to 1mg/ml by addition of sterile water and the pH of the mixture adjusted to4 with several drops of 0.5M acetic acid.

MePEG-MGDF conjugate was separated from the excess of MePEG and otherreaction by-products by ion-exchange chromatography using SP SepharoseHP (Pharmacia Biotech) ion exchange resin.

The reaction mixture was loaded (2.5 mg/ml of resin) onto the column andthe unreacted MePEG was eluted with 3 column volumes of the startingbuffer A (20 mM sodium phosphate, pH 7.2, 15% glycerol). After that, theMePEG-MGDF conjugate was eluted using a linear gradient from 0% to 30%in 10 column volumes of the end buffer B (1M NaCl in buffer A). Theeluent was monitored at 280 nm. Fractions containing poly-MePEG-MGDFconjugate were pooled, concentrated and sterile filtered.

The purified poly-MePEG-MGDF conjugate was analyzed by HPLC SEC usingTSK-GEL G4000SWXL and G2000SWXL gel filtration columns coupled inseries. Proteins were detected by UV absorbance at 280 nm. BIO-RAD gelfiltration standards served as globular protein molecular weightmarkers.

As can be seen in FIG. 17A, HPLC SEC reveals two major components in thepreparation (in about a 2 to 1 ratio) elution positions of whichcorrespond to those of 370.9 kDa and 155.0 kDa globular proteinsrespectively. See also Table 8 below.

Conjugates PEG 9, PEG 10 and PEG 12 prepared by MGDF acylation withsuccinimidyl esters of MW=6-50 kDa MePEGs were conducted similarly. Themajor reaction parameters used in these preparations are summarizable inTable 7.

Results of HPLC SEC analyses of these conjugates are shown in Table 8.

12.2. Preparation of poly-MePEG-MGDF conjugates by MGDF reductivealkylation with MePEG aldehydes

Preparation of poly-MePEG(20 kDa)-MGDF conjugate (PEG 20).

To a cooled (4° C.), stirred solution of MGDF (2 ml, 2.5 mg/ml) in 100mM sodium phosphate, pH 5, containing 20 mM NaCNBH3 was added a 10-foldmolar excess of monomethoxy-polyethylene glycol aldehyde (MePEG)(average molecular weight 20 kDa) and the stirring of the reactionmixture was continued at the same temperature.

The extent of the protein modification during the course of the reactionwas monitored by SEC HPLC using Superdex 200 HR 10/30 column (PharmaciaBiotech) eluted with 0.1M sodium phosphate buffer pH 6.9 at 0.7 ml/min.

After 16 hours the SEC HPLC analysis indicated that more than 90% of theinitial amount of the protein has been modified. At this time theprotein concentration in the reaction mixture was brought to 1 mg/ml bydilution of the reaction mixture with sterile water and the pH adjustedto 4 (0.5M acetic acid).

MePEG-MGDF conjugate was separated from the excess of MePEG and otherreaction by-products by ion-exchange chromatography using SP SepharoseHP (Pharmacia Biotech) ion exchange resin.

The reaction mixture was loaded (2.5 mg/ml of resin) onto the column andthe unreacted MePEG was eluted with 3 column volumes of the startingbuffer A (20 mM sodium phosphate, pH 7.2, 15% glycerol). After that, theMePEG-MGDF conjugate was eluted using a linear gradient from 0% to 30%in 10 column volumes of the end buffer B (1M NaCl in buffer A). Theeluent was monitored at 280 nm. Fractions containing poly-MePEG-MGDFconjugate were pooled, concentrated and sterile filtered.

The purified poly-MePEG-MGDF conjugate was analyzed by HPLC SEC usingTSK-GEL G4000SWXL and G2000SWXL gel filtration columns coupled inseries. Proteins were detected by UV absorbance at 280 nm. BIO-RAD gelfiltration standards served as globular protein molecular weightmarkers.

As can be seen in FIG. 17B, HPLC SEC reveals two major components(constituting 52% and 47% of the total amount) in the preparation,elution positions of which correspond to those of 359.4 kDa and 159.3kDa globular proteins respectively. See also Table 8.

Conjugates PEG 18, PEG 19 and PEG 21 prepared by MGDF reductivealkylation with MePEG aldehydes of MW=6-25 kDa were conducted similarly.The major reaction parameters used in these preparations are summarizedin Table 7.

Results of HPLC SEC analyses of these conjugates are shown in Table 8.

12.3. Preparation of monomethoxy-polyethylene glycol-MGDF conjugateswith the site of attachment at the N-terminal α-amino residue

Preparation of mono-MePEG (20 kDa)-MGDF conjugate (PEG 16)

To a cooled (4° C.), stirred solution of MGDF (2 ml, 2.5 mg/ml) in 100mM sodium phosphate, pH 5, containing 20 mM NaCNBH3 was added a 5-foldmolar excess of methoxypolyethylene glycol aldehyde (MePEG) (averagemolecular weight 20 kDa) and the stirring of the reaction mixture wascontinued at the same temperature.

The extent of the protein modification during the course of the reactionwas monitored by SEC HPLC using Superdex 200 HR 10/30 column (PharmaciaBiotech) eluted with 0.1M sodium phosphate buffer pH 6.9 at 0.7 ml/min.

After 16 hours the SEC HPLC analysis indicated that about 90% of theinitial amount of the protein has been modified. At this time theprotein concentration in the reaction mixture was reduced to 1 mg/ml bydilution with sterile water and the pH of the reaction mixture adjustedto 4 (0.5M acetic acid).

The mono-MePEG (20 kDa)-MGDF conjugate was separated from the excess ofMePEG and other reaction by-products by ion-exchange chromatographyusing SP Sepharose HP (Pharmacia Biotech) ion exchange resin.

The reaction mixture was loaded (2.5 mg/ml of resin) onto the column andthe unreacted MePEG was eluted with 3 column volumes of the startingbuffer A (20 mM sodium phosphate, pH 7.2, 15% glycerol). After that, theMePEG-MGDF conjugate was eluted using a linear gradient from 0% to 25%of the end buffer B (1M NaCl in buffer A) in 20 column volumes. Theeluent was monitored at 280 nm. Fractions containing poly-MePEG-MGDFconjugate were pooled, concentrated and sterile filtered.

The homogeneity of the mono-MePEG-MGDF conjugates was determined bySodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis using 4-20%precast gradient gels (NOVEX). One major band corresponding to theposition of a 46.9 kDa protein was revealed.

The purified poly-MePEG-MGDF conjugate was analyzed by HPLC SEC usingTSK-GEL G4000SWXL and G2000SWXL gel filtration columns coupled inseries. Proteins were detected by UV absorbance at 280 nm. The BIO-RADgel filtration standards served as globular protein molecular weightmarkers.

As can be seen in FIG. 17C, SEC HPLC reveals one major component in thepreparation, elution positions of which corresponds to that of 181.1 kDaglobular protein. See also Table 9.

Mono-MePEG-MGDF conjugates PEG 14, PEG 15 and PEG 17 prepared by MGDFreductive alkylation with MePEG aldehydes of MW=6-25 kDa were conductedsimilarly. The major reaction parameters used in these preparations aresummarized in Table 7.

Results of HPLC SEC analyses of these conjugates are shown in Table 9.

                  TABLE 7                                                         ______________________________________                                        Summary of MGDF modification reaction parameters                                           Reaction conditions                                                                                          Molar                                                    MGDF      Temp-      Ratio                                   Reactive MePEG   conc.     erature,                                                                            Time,                                                                              MePEG/                            Code  Type     MW      mg/ml pH  °C                                                                           h    MGDF                              ______________________________________                                        PEG 9 NHS       6kDa   2.5   8   r.t.  0.5  15                                      ester                                                                   PEG 10                                                                              NHS       6kDa   2.5   8   r.t.  0.5  10                                      ester                                                                   PEG 11                                                                              NHS      20kDa   2.5   8   r.t.  0.5  10                                      ester                                                                   PEG 12                                                                              NHS      50kDa   2.5   8   r.t.  0.5   5                                      ester                                                                   PEG 14                                                                              ALDE-     6 kDa  2.5   5   4° C.                                                                        16    5                                      HYDE                                                                    PEG 15                                                                              ALDE-    12kDa   2.5   5   4° C.                                                                        16    5                                      HYDE                                                                    PEG 16                                                                              ALDE-    20kDa   2.5   5   4° C.                                                                        16    5                                      HYDE                                                                    PEG 17                                                                              ALDE-    25kDa   2.5   5   4° C.                                                                        16   10                                      HYDE                                                                    PEG 18                                                                              ALDE-     6kDa   5     5   4° C.                                                                        16   10                                      HYDE                                                                    PEG 19                                                                              ALDE-    12kDa   5     5   4° C.                                                                        16   10                                      HYDE                                                                    PEG 20                                                                              ALDE-    20kDa   5     5   4° C.                                                                        16   10                                      HYDE                                                                    PEG 21                                                                              ALDE-    25kDa   5     5   4° C.                                                                        16   10                                      HYDE                                                                    ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Summary of poly-MePEG-MGDF characteristics by SEC HPLC                                                  Apparent MW by                                                                          Component                                 Code   Reactive   MePEG   SEC, kDa  amount, %                                 ______________________________________                                        PEG 9  NHS         6kDa    87.9     75                                               ester               52.7     25 (shoulder)                             PEG 10 NHS         6kDa    69.2     14 (shoulder)                                    ester               42.9     86                                        PEG 11 NHS        20kDa   370.9     68                                               ester              155.0     32                                        PEG 12 NHS        50kDa   865.6     53                                               ester              368.0     47                                        PEG 18 ALDEHYDE    6kDa    84.6     60                                                                   41.5     40                                        PEG 19 ALDEHYDE   12kDa   218.4     59                                                                  106.7     41                                        PEG 20 ALDEHYDE   20kDa   359.4     52                                                                  159.3     47                                        PEG 21 ALDEHYDE   25kDa   450.5     54                                                                  218.4     46                                        ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Apparent molecular weights of mono-MePEG-MGDF Conjugates                            Reactive   MePEG   Apparent MW by                                                                          Apparent MW by                             Code  Type       MW      SEC, kDa  SDS PAGE, kDa                              ______________________________________                                        PEG 14                                                                              ALDEHYDE    6 kDa   44.5     27.7                                       PEG 15                                                                              ALDEHYDE   12kDa   104.7     38.3                                       PEG 16                                                                              ALDEHYDE   20kDa   181.1     46.9                                       PEG 17                                                                              ALDEHYDE   25kDa   226.4     55.5                                       ______________________________________                                    

EXAMPLE 13 Biological Activity of Pegylated MGDF Molecules

Platelet counts from mice treated with recombinant human MGDF weremeasured and the results are presented in FIG. 18. CHO-derived 1-332MGDF (MGDF-1; amino acids 1-332 of SEQ ID NO: 25) (open diamond),unpegylated E. coli 1-63 MGDF (MGDF-11; amino acids 1-163 of SEQ ID NO:25) (open circles) and pegylated E. coli 1-63 MGDF (MGDF-11; amino acids1-163 of SEQ ID NO: 25) (closed circles) MGDF at the indicatedconcentrations were injected subcutaneously into normal Balb/c mice oncedaily for 5 days. Test bleeds from a small lateral cut in a tail veinwere collected 24 hours after the last injection. Blood cell analyseswere performed with a Sysmex electronic blood cell analyser (BaxterDiagnostics, Inc. Irvine, Calif.). Data are represented as the mean ofdeterminations of 4 animals, ± standard error of the mean. Other bloodcell parameters such as total white blood cell counts or red blood cellcounts were not affected by this treatment.

Additional forms of recombinant human MGDF were tested as above.Platelet counts from mice treated with either 50 ug/kg/day or 10ug/kg/day of the indicated form of r-HuMGDF are shown in the followingTable 10. Data are the mean of 4 animals and the standard errors areitalicized.

                  TABLE 10                                                        ______________________________________                                                  50 ug/kg/day     10 ug/kg/day                                       Form        Mean (n = 4)                                                                            sem      Mean (n = 4)                                                                          sem                                    ______________________________________                                        CHO MGOF-1  4343      309      2571    80                                     (amino acids 1-332                                                            of SEQ ID NO:1)                                                               E. Coli 22 184                                                                            2021       29      1439    18                                     PEG 9       2728       56      2369    34                                     PEG 10      2431      291      1556    126                                    PEG 11      3778       59      1861    73                                     PEG 12      3885      156      1740                                           PEG 14      3567       80      2020    63                                     PEG 15      4402       57      2834    99                                     PEG 16      4511      239      3215    11                                     PEG 17      4140      188      3113    261                                    PEG 18      4586       59      2931    129                                    PEG 19      3980      330      4189    80                                     PEG 20      3942      285      3054    339                                    PEG 21      4195      145      4002    91                                     Baseline     939       25                                                     ______________________________________                                    

Key to Table 10

In each of the following, the MGDF molecule that was pegylated was E.coli derived MGDF-11 (amino acids, numbering from the beginning of themature protein), as described in the above Example 12:

    ______________________________________                                                                       Reactive PEG                                                       Avg. MW    molecule for                                   Name     Pegylation of PEG     synthesis                                      ______________________________________                                        PEG 9    polypegylated                                                                             6 kDa     NHS ester of MePEG                             PEG 10   polypegylated                                                                             6 kDa     NHS ester of MePEG                             PEG 11   polypegylated                                                                            20 kDa     NHS ester of MePEG                             PEG 12   polypegylated                                                                            50 kDa     NHS ester of MePEG                             PEG 14   monopegylated                                                                             6 kDa     Aldehyde of MePEG                              PEG 15   monopegylated                                                                            12 kDa     Aldehyde of MePEG                              PEG 16   monopegylated                                                                            20 kDa     Aldehyde of MePEG                              PEG 17   monopegylated                                                                            25 kDa     Aldehyde of MePEG                              PEG 18   polypegylated                                                                             6 kDa     Aldehyde of MePEG                              PEG 19   polypegylated                                                                            12 kDa     Aldehyde of MePEG                              PEG 20   polypegylated                                                                            20 kDa     Aldehyde of MePEG                              PEG 21   polypegylated                                                                            25 kDa     Aldehyde of MePEG                              ______________________________________                                    

The baseline counts are in normal animals without administration of anymaterials.

It is clear that pegylation of recombinant human MGDF does not adverselyaffect the ability of the molecule to increase platelet counts inrecipient animals, and may in fact increase the activity of the E. coliproduct 1-63 MGDF (MGDF-11; amino acids 1-163 of SEQ ID NO: 25) to be asgreat or greater than that seen with the CHO-derived 1-332 MGDF (MGDF-1;amino acids 1-332 of SEQ ID NO: 25) molecule.

While the present invention has been described above both generally andin terms of preferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art in light of theabove description. Therefore, it is intended that the appended claimscover all such variations coming within the scope of the invention asclaimed.

Additionally, the publications and other materials cited to illuminatethe background of the invention, and in particular cases to provideadditional details concerning its practice, are herein incorporated byreference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 27                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaProProAlaXaaAspProArgLeuLeuAsnLysMetLeuArgAsp                              151015                                                                        SerHisValLeuHisXaaArgLeuXaaGlnXaaProAspIleTyr                                 202530                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaProProAlaXaaAspProArgLeuLeuAsnLysMetLeuArgAsp                              151015                                                                        SerHisValLeuHis                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ThrGlnLysGluGlnThrLysAlaGlnAspValLeuGlyAlaValAla                              151015                                                                        Leu                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCNCCNCCNGCNTGYGA17                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCARTGYAACACRTGNGARTC21                                                       (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAsp                              151015                                                                        SerHisValLeuHis                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GTACGCGTTCTAGANNNNNNT21                                                       (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AGTTTACTGAGGACTCGGAGG21                                                       (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TTCGGCCGGATAGGCCTTTTTTTTTTTTTT30                                              (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TTCGGCCGGATAGGCCTTTTTTTTTTTTT29                                               (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TGCGACCTCCGAGTCCTCAG20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GAGTCCTCAGTAAACTGCTTCGT23                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGAGTCACGAAGCAGTTTAC20                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCTTTACTTCTAGGCCTG18                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GAGGTCACAAGCAGGAGGA19                                                         (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGCATAGTCCGGGACGTCG19                                                         (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TCCTCCTGCTTGTGACCTC19                                                         (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CCAGGAAGGATTCAGGGGA19                                                         (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CAACAAGTCGACCGCCAGCCAGACACCCCG30                                              (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GGCCGGATAGGCCACTCNNNNNNT24                                                    (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GCARTGYAANACRTGNGARTC21                                                       (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      TTGGTGTGCACTTGTG16                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CACAAGTGCACACCAACCCC20                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1342 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 36..1097                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 99..1097                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 36..98                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      CAGGGAGCCACGCCAGCCAAGACACCCCGGCCAGAATGGAGCTGACTGAATTG53                       MetGluLeuThrGluLeu                                                            21- 20                                                                        CTCCTCGTGGTCATGCTTCTCCTAACTGCAAGGCTAACGCTGTCCAGC101                           LeuLeuValValMetLeuLeuLeuThrAlaArgLeuThrLeuSerSer                              15-10-51                                                                      CCGGCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGT149                           ProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArg                              51015                                                                         GACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCAC197                           AspSerHisValLeuHisSerArgLeuSerGlnCysProGluValHis                              202530                                                                        CCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGA245                           ProLeuProThrProValLeuLeuProAlaValAspPheSerLeuGly                              354045                                                                        GAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGA293                           GluTrpLysThrGlnMetGluGluThrLysAlaGlnAspIleLeuGly                              50556065                                                                      GCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG341                           AlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGlnLeu                              707580                                                                        GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTC389                           GlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnVal                              859095                                                                        CGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCT437                           ArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuPro                              100105110                                                                     CCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTG485                           ProGlnGlyArgThrThrAlaHisLysAspProAsnAlaIlePheLeu                              115120125                                                                     AGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA533                           SerPheGlnHisLeuLeuArgGlyLysValArgPheLeuMetLeuVal                              130135140145                                                                  GGAGGGTCCACCCTCTGCGTCAGGCGGGCCCCACCCACCACAGCTGTC581                           GlyGlySerThrLeuCysValArgArgAlaProProThrThrAlaVal                              150155160                                                                     CCCAGCAGAACCTCTCTAGTCCTCACACTGAACGAGCTCCCAAACAGG629                           ProSerArgThrSerLeuValLeuThrLeuAsnGluLeuProAsnArg                              165170175                                                                     ACTTCTGGATTGTTGGAGACAAACTTCACTGCCTCAGCCAGAACTACT677                           ThrSerGlyLeuLeuGluThrAsnPheThrAlaSerAlaArgThrThr                              180185190                                                                     GGCTCTGGGCTTCTGAAGTGGCAGCAGGGATTCAGAGCCAAGATTCCT725                           GlySerGlyLeuLeuLysTrpGlnGlnGlyPheArgAlaLysIlePro                              195200205                                                                     GGTCTGCTGAACCAAACCTCCAGGTCCCTGGACCAAATCCCCGGATAC773                           GlyLeuLeuAsnGlnThrSerArgSerLeuAspGlnIleProGlyTyr                              210215220225                                                                  CTGAACAGGATACACGAACTCTTGAATGGAACTCGTGGACTCTTTCCT821                           LeuAsnArgIleHisGluLeuLeuAsnGlyThrArgGlyLeuPhePro                              230235240                                                                     GGACCCTCACGCAGGACCCTAGGAGCCCCGGACATTTCCTCAGGAACA869                           GlyProSerArgArgThrLeuGlyAlaProAspIleSerSerGlyThr                              245250255                                                                     TCAGACACAGGCTCCCTGCCACCCAACCTCCAGCCTGGATATTCTCCT917                           SerAspThrGlySerLeuProProAsnLeuGlnProGlyTyrSerPro                              260265270                                                                     TCCCCAACCCATCCTCCTACTGGACAGTATACGCTCTTCCCTCTTCCA965                           SerProThrHisProProThrGlyGlnTyrThrLeuPheProLeuPro                              275280285                                                                     CCCACCTTGCCCACCCCTGTGGTCCAGCTCCACCCCCTGCTTCCTGAC1013                          ProThrLeuProThrProValValGlnLeuHisProLeuLeuProAsp                              290295300305                                                                  CCTTCTGCTCCAACGCCCACCCCTACCAGCCCTCTTCTAAACACATCC1061                          ProSerAlaProThrProThrProThrSerProLeuLeuAsnThrSer                              310315320                                                                     TACACCCACTCCCAGAATCTGTCTCAGGAAGGGTAAGGTTCTCAGA1107                            TyrThrHisSerGlnAsnLeuSerGlnGluGly*                                            325330                                                                        CACTGCCGACATCAGCATTGTCTCGTGTACAGCTCCCTTCCCTGCAGGGCGCCCCTGGGA1167              GACAACTGGACAAGATTTCCTACTTTCTCCTGAAACCCAAAGCCCTGGTAAAAGGGATAC1227              ACAGGACTGAAAAGGGAATCATTTTTCACTGTACATTATAAACCTTCAGAAGCTATTTTT1287              TTAAGCTATCAGCAATACTCATCAGAGCAGCTAGCTCTTTGGTCTATTTTCTGCA1342                   (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 353 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      MetGluLeuThrGluLeuLeuLeuValValMetLeuLeuLeuThrAla                              21-20-15-10                                                                   ArgLeuThrLeuSerSerProAlaProProAlaCysAspLeuArgVal                              51510                                                                         LeuSerLysLeuLeuArgAspSerHisValLeuHisSerArgLeuSer                              152025                                                                        GlnCysProGluValHisProLeuProThrProValLeuLeuProAla                              303540                                                                        ValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLys                              455055                                                                        AlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMet                              60657075                                                                      AlaAlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGly                              808590                                                                        GlnLeuSerGlyGlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeu                              95100105                                                                      LeuGlyThrGlnLeuProProGlnGlyArgThrThrAlaHisLysAsp                              110115120                                                                     ProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysVal                              125130135                                                                     ArgPheLeuMetLeuValGlyGlySerThrLeuCysValArgArgAla                              140145150155                                                                  ProProThrThrAlaValProSerArgThrSerLeuValLeuThrLeu                              160165170                                                                     AsnGluLeuProAsnArgThrSerGlyLeuLeuGluThrAsnPheThr                              175180185                                                                     AlaSerAlaArgThrThrGlySerGlyLeuLeuLysTrpGlnGlnGly                              190195200                                                                     PheArgAlaLysIleProGlyLeuLeuAsnGlnThrSerArgSerLeu                              205210215                                                                     AspGlnIleProGlyTyrLeuAsnArgIleHisGluLeuLeuAsnGly                              220225230235                                                                  ThrArgGlyLeuPheProGlyProSerArgArgThrLeuGlyAlaPro                              240245250                                                                     AspIleSerSerGlyThrSerAspThrGlySerLeuProProAsnLeu                              255260265                                                                     GlnProGlyTyrSerProSerProThrHisProProThrGlyGlnTyr                              270275280                                                                     ThrLeuPheProLeuProProThrLeuProThrProValValGlnLeu                              285290295                                                                     HisProLeuLeuProAspProSerAlaProThrProThrProThrSer                              300305310315                                                                  ProLeuLeuAsnThrSerTyrThrHisSerGlnAsnLeuSerGlnGlu                              320325330                                                                     Gly                                                                           (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1164 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 34..894                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 97..894                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 34..96                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AGGGAGCCACGCCAGCCAGACACCCCGGCCAGAATGGAGCTGACTGAATTGCTC54                      MetGluLeuThrGluLeuLeu                                                         21-20- 15                                                                     CTCGTGGTCATGCTTCTCCTAACTGCAAGGCTAACGCTGTCCAGCCCG102                           LeuValValMetLeuLeuLeuThrAlaArgLeuThrLeuSerSerPro                              10-51                                                                         GCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGAC150                           AlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAsp                              51015                                                                         TCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCT198                           SerHisValLeuHisSerArgLeuSerGlnCysProGluValHisPro                              202530                                                                        TTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAA246                           LeuProThrProValLeuLeuProAlaValAspPheSerLeuGlyGlu                              35404550                                                                      TGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCA294                           TrpLysThrGlnMetGluGluThrLysAlaGlnAspIleLeuGlyAla                              556065                                                                        GTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA342                           ValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGlnLeuGly                              707580                                                                        CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGT390                           ProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArg                              859095                                                                        CTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCA438                           LeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProPro                              100105110                                                                     CAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGC486                           GlnGlyArgThrThrAlaHisLysAspProAsnAlaIlePheLeuSer                              115120125130                                                                  TTCCAACACCTGCTCCGAGGAAAGGACTTCTGGATTGTTGGAGACAAA534                           PheGlnHisLeuLeuArgGlyLysAspPheTrpIleValGlyAspLys                              135140145                                                                     CTTCACTGCCTCAGCCAGAACTACTGGCTCTGGGCTTCTGAAGTGGCA582                           LeuHisCysLeuSerGlnAsnTyrTrpLeuTrpAlaSerGluValAla                              150155160                                                                     GCAGGGATTCAGAGCCAAGATTCCTGGTCTGCTGAACCAAACCTCCAG630                           AlaGlyIleGlnSerGlnAspSerTrpSerAlaGluProAsnLeuGln                              165170175                                                                     GTCCCTGGACCAAATCCCCGGATACCTGAACAGGATACACGAACTCTT678                           ValProGlyProAsnProArgIleProGluGlnAspThrArgThrLeu                              180185190                                                                     GAATGGAACTCGTGGACTCTTTCCTGGACCCTCACGCAGGACCCTAGG726                           GluTrpAsnSerTrpThrLeuSerTrpThrLeuThrGlnAspProArg                              195200205210                                                                  AGCCCCGGACATTTCCTCAGGAACATCAGACACAGGCTCCCTGCCACC774                           SerProGlyHisPheLeuArgAsnIleArgHisArgLeuProAlaThr                              215220225                                                                     CAACCTCCAGCCTGGATATTCTCCTTCCCCAACCCATCCTCCTACTGG822                           GlnProProAlaTrpIlePheSerPheProAsnProSerSerTyrTrp                              230235240                                                                     ACAGTATACGCTCTTCCCTCTTCCACCCACCTTGCCCACCCCTGTGGT870                           ThrValTyrAlaLeuProSerSerThrHisLeuAlaHisProCysGly                              245250255                                                                     CCAGCTCCACCCCCTGCTTCCTGACCCTTCTGCTCCAACGCCCACCCCTACCAG924                     ProAlaProProProAlaSer*                                                        260265                                                                        CCCTCTTCTAAACACATCCTACACCCACTCCCAGAATCTGTCTCAGGAAGGGTAAGGTTC984               TCAGACACTGCCGACATCAGCATTGTCTCGTGTACAGCTCCCTTCCCTGCAGGGCGCCCC1044              TGGGAGACAACTGGACAAGATTTCCTACTTTCTCCTGAAACCCAAAGCCCTGGTAAAAGG1104              GATACACAGGACTGAAAAGGGAATCATTTTTCACTGTACATTATAAACCTTCAGAAGCTA1164              (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 286 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      MetGluLeuThrGluLeuLeuLeuValValMetLeuLeuLeuThrAla                              21-20-15-10                                                                   ArgLeuThrLeuSerSerProAlaProProAlaCysAspLeuArgVal                              51510                                                                         LeuSerLysLeuLeuArgAspSerHisValLeuHisSerArgLeuSer                              152025                                                                        GlnCysProGluValHisProLeuProThrProValLeuLeuProAla                              303540                                                                        ValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLys                              455055                                                                        AlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMet                              60657075                                                                      AlaAlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGly                              808590                                                                        GlnLeuSerGlyGlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeu                              95100105                                                                      LeuGlyThrGlnLeuProProGlnGlyArgThrThrAlaHisLysAsp                              110115120                                                                     ProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysAsp                              125130135                                                                     PheTrpIleValGlyAspLysLeuHisCysLeuSerGlnAsnTyrTrp                              140145150155                                                                  LeuTrpAlaSerGluValAlaAlaGlyIleGlnSerGlnAspSerTrp                              160165170                                                                     SerAlaGluProAsnLeuGlnValProGlyProAsnProArgIlePro                              175180185                                                                     GluGlnAspThrArgThrLeuGluTrpAsnSerTrpThrLeuSerTrp                              190195200                                                                     ThrLeuThrGlnAspProArgSerProGlyHisPheLeuArgAsnIle                              205210215                                                                     ArgHisArgLeuProAlaThrGlnProProAlaTrpIlePheSerPhe                              220225230235                                                                  ProAsnProSerSerTyrTrpThrValTyrAlaLeuProSerSerThr                              240245250                                                                     HisLeuAlaHisProCysGlyProAlaProProProAlaSer                                    255260265                                                                     __________________________________________________________________________

What is claimed is:
 1. A polypeptide consisting of amino acids 1-163 ofSEQ ID NO: 25, wherein a single polyethylene glycol is attached to theα-amino group at the N-terminus of said polypeptide.
 2. A polypeptideaccording to claim 1, wherein said polyethylene glycol has an averagemolecular weight of 5 kDa to 50 kDa.
 3. A polypeptide according to claim1, produced by a process comprising the following steps:a) reacting apolypeptide precursor consisting of amino acids 1-163 of SEQ ID NO:25with a polyethylene glycol molecule having a single reactive aldehydegroup under reductive alkylation conditions, at a pH sufficiently acidicto allow the α-amino group at the amino terminus of said polypeptideprecursor to be reactive; and b) obtaining a monopegylated polypeptideproduct.
 4. A polypeptide according to claim 3, wherein said reductivealkylation conditions involve the use of sodium cyanoborohydride as areducing agent.
 5. A substantially homogeneous preparation of apolypeptide consisting of amino acids 1-163 of SEQ ID NO: 25monopegylated on the α-amino group at the N-terminus of saidpolypeptide.
 6. A pharmaceutical composition comprising a polypeptideaccording to any of claims 1, 2, 3, 4, or 5 and a pharmaceuticallyacceptable diluent, adjuvant or carrier.
 7. A pharmaceutical compositionaccording to claim 6, comprising an aqueous solution.
 8. Apharmaceutical composition according to claim 6, wherein saidcomposition is in the form of a lyophilizate.
 9. A pharmaceuticalcomposition comprising:(a) a substantially homogeneous preparation of apolypeptide consisting of amino acids 1-163 of SEQ ID NO:25monopegylated on the α-amino group at the N-terminus of said polypeptidevia an alkyl linkage, wherein the polyethylene glycol has a molecularweight of 5 kDa to 50 kDa; and (b) a pharmaceutically acceptablediluent, adjuvant or carrier.
 10. A pharmaceutical composition accordingto claim 9, comprising an aqueous solution.
 11. A pharmaceuticalcomposition according to claim 10, wherein said composition is in theform of a lyophilizate.